Carbopyronone Compounds Useful as Diagnostic Adjuvants

ABSTRACT

Described herein are fluorescent compounds and methods and comprising these compounds. The compounds disclosed herein are carbopyronine reagents that fluoresce in the red portion of the UV/VIS spectrum and provide bright fluorescence intensity, uniform cell staining, and good retention within live cells as well as low toxicity toward cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. Non-Provisional patentapplication Ser. No. 16/002,991, filed Jun. 7, 2018, which is aDivisional of U.S. Non Provisional patent application Ser. No.15/304,800, filed Oct. 17, 2016, now U.S. Pat. No. 10,018,617 issued onJul. 10, 2018, which is a 371 National Stage of PCT/US2015/030956, filedon May 15, 2015, now expired, which claims the benefit of U.S.Provisional Patent Application No. 61/994,555, filed May 16, 2014, nowexpired, which are herein incorporated by reference in their entireiesentireties.

FIELD

The present disclosure relates to carbopyronine fluorescent dyecompounds, including reactive dye derivatives and dye-conjugates, andtheir uses as cell tracking reagents as well as in staining samples,labeling proteins, detecting ligands or other analytes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 8, 2015, isnamed LT00888PCT_SL.txt and is 468 bytes in size.

BACKGROUND

Methods for monitoring cell proliferation, differentiation, and functionusing flow cytometry have enabled investigation of complex biologicalphenomena, e.g., immune responses to antigen, which responses involvecomplex interactions among multiple cell types (see, Wallace et al.,Cytometry Part A 73A: 1019-1034 (2008)). So called cell-tracking dyeshave proven useful for qualitative and quantitative monitoring of celldivision, both in vivo and in vitro (see, Hawkins et al., Nat Protoc2:2057-2067 (2007); and Wallace et al., Immunol Invest 36:527-561,(2007)). These dyes, also referred to herein as cell-tracking reagentsor cell-tracking compounds which term is inclusive of cell-tracingreagents and cell-tracing compounds, generate a fluorescent signal that,while relatively stable in non-dividing cells, progressively decreaseswith each round of cell division. Reduction in fluorescence intensitycan be quantified by flow cytometry in conjunction with any of severaldifferent algorithms to estimate the extent of proliferation (inresponse to a particular stimulus) based on dye dilution (see, Wallaceet al., Cytometry Part A 73A: 1019-1034 (2008)). A major advantage ofusing flow cytometry in conjunction with cell-tracking reagents tomonitor the extent of cell division is that cells can also be stainedfor expression of other cell surface or intracellular markers to definelineage, functionality, activation state, cytokine expression, etc.(see, Bercovici et al., J Immunol Methods 276:5-17, (2003); Fazekas deSt Groth et al., Immunol Cell Biol 77:530-538, (1999); and Tanaka etal., Immunol Invest 33:309-324, (2004)).

Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE or,alternatively, CFSE) remains a popular, commercially availablecell-tracking reagent, excitable with 488-nm laser light to give abright green fluorescence. CFDA-SE has been widely used to monitor cellproliferation by flow cytometry in heterogeneous cell populations andstains cells with a bright homogeneous fluorescence, which ispartitioned between daughter cells during each cell division.

Notwithstanding the current popularity of CFDA-SE, there remains a needfor alternative fluorescent dyes, useful as cell-tracking reagents, withdifferent spectral properties. Such reagents may be combined forsimultaneous use with other currently-available cell analysis reagents,such as, for example, the 488 nm-excitable reagent Green FluorescentProtein (GFP), or with the 405 nm-excitable violet-emitting dye PACIFICBLUE (Thermo Fisher Scientific), thereby permitting researchers to studycell proliferation, differentiation, and/or function in otherwiseindistinguishable cell populations in mixed cell cultures withmulti-color applications using flow cytometry.

The development of carbopyronine-based cell-tracking reagents thatfluoresce in the red portion of the UV/VIS spectrum to provide brightfluorescence intensity for long-term monitoring of cell proliferation,differentiation, migration, location, and/or function using flowcytometry and/or fluorescence microscopy has, heretofore, not beenrealized.

SUMMARY

Described herein are dye compounds, methods, and kits that may be usedto stain samples, label proteins, detect ligands or other analytes, andfor short- or long-term tracking of cell proliferation, differentiation,structure and/or function. The compounds disclosed herein arecarbopyronine-based dye compounds that fluoresce in the red portion ofthe UV/VIS spectrum and provide bright fluorescence intensity, uniformcell staining, good retention within cells and have low toxicity towardcells. These compounds may be used in place of and/or in combinationwith other currently-available cell analysis reagents, such as, forexample, the 488 nm-excitable Green Fluorescent Protein (GFP), to trackand/or stain otherwise indistinguishable cell populations in mixed cellcultures via flow cytometry and/or fluorescence microscopy,respectively.

One illustrative embodiment provides a compound having structuralformula (I):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵, and R¹⁶ are eachindependently H, halogen, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c); or

R⁷ taken together with R¹⁴ are part of an optionally substituted 5- or6-membered ring;

R⁸ taken together with R¹⁵ are part of an optionally substituted 5- or6-membered ring;

R⁹ taken together with R¹¹ are part of an optionally substituted 5- or6-membered ring;

R¹⁰ taken together with R¹⁶ are part of an optionally substituted 5- or6-membered ring;

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance.

In certain embodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). Incertain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are each independently H,alkyl, substituted alkyl, or sulfoalkyl. In certain embodiments, R⁷, R⁸,R⁹, and R¹⁰ are each independently a C₁-C₆ alkyl, which can be the sameor different. In certain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are eachmethyl. In certain embodiments, R¹² and R¹³ are each alkyl, preferably aC₁-C₆ alkyl. In certain embodiments, R¹² and R¹³ are each methyl. Incertain embodiments, R¹¹ and R¹⁴ are each independently alkyl,substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certainembodiments, R¹¹ and R¹⁴ are each independently a C₁-C₆ alkyl, a C₁-C₆substituted alkyl, or sulfoalkyl, which can be the same or different. Incertain embodiments, R_(x) is succinimidyl ester (SE),sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP) ester,tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another illustrative embodiment provides a compound having structuralformula (II):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹ and R²²are each independently H, halogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R³, R⁴, R⁵ and R⁶ are each independently H or halo. Incertain embodiments, R³, R⁴, R⁵ and R⁶ are independently H, Cl, or F. Incertain embodiments, R⁷ and R⁹ are each independently alkyl, substitutedalkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certain embodiments,R⁷ and R⁹ are each independently methyl, -L-R_(x), -L-S_(c) or(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 3. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another illustrative embodiment provides a compound having structuralformula (III):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³,and R²⁴ are each independently H, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R⁷, R⁹, R¹⁷, R¹⁹, R²⁰, R²², R²³ and R²⁴ are eachindependently alkyl, substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or-L-S_(c). In certain embodiments, R⁷, R⁹, R¹⁷, R²², R²³ and R²⁴ are eachmethyl. In certain embodiments, R¹⁹ and R²⁰ are each independently(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 1. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another illustrative embodiment provides a compound having structuralformula (IV):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³,and R²⁴ are each independently H, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance.

In certain embodiments, R⁹, R¹², R¹³, R²² and R²³ are each alkyl,preferably a C₁-C₆ alkyl. In certain embodiments, R⁹, R¹², R¹³, R²² andR²³ are each methyl. In certain embodiments, R³, R⁴, R⁵ and R⁶ are eachindependently H or halo. In certain embodiments, R³ and R⁶ areindependently H, Cl, or F. In certain embodiments, R² is carboxyl. Incertain embodiments, R⁷, R⁹ and R²⁰ are each independently alkyl,substituted alkyl, sulfoalkyl, sulfo, (CH₂)_(n)SO₃ ⁻, wherein n is aninteger between 1 and 6, -L-R_(x), or -L-S_(c). In certain embodiments,R⁷ is -L-R_(x) and R²⁰ is (CH₂)_(n)SO₃ ⁻. In certain embodiments, nis 1. In certain embodiments, R_(x) is succinimidyl ester (SE),sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP) ester,tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

In certain embodiments, R_(x) is selected from an acrylamide, anactivated ester of a carboxylic acid, a carboxylic ester, an acyl azide,an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline,an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a photoactivatable group, a reactive platinum complex,a silyl halide, a sulfonyl halide, and a thiol. In certain embodimentsthe reactive group is selected from the group consisting of carboxylicacid, succinimidyl ester of a carboxylic acid, hydrazide, amine and amaleimide. The reactive group may be attached to any appropriate site onthe reporter molecule or the aniline moiety. In certain embodiments, atleast one member selected from R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹²,R¹³, R¹⁹ and R²⁰ is a reactive group. In certain embodiments, at leastone of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is a reactive group. Incertain embodiments, at least one of R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is areactive group.

In certain embodiments, S_(c) is selected from a carrier molecule and asolid support. In certain embodiments, S_(c) is selected from an aminoacid, a polymer of amino acids, a peptide, a protein, a neurotoxin, aphallotoxin, a cytokine, a toxin, a protease substrate, a protein kinasesubstrate, an enzyme, an antibody, an antibody fragment, a lectin, aglycoprotein, a histone, an albumin, a lipoprotein, avidin,streptavidin, protein A, protein G, a phycobiliprotein, a fluorescentprotein, a hormone, a growth factor, a nucleic acid base, a nucleoside,a nucleotide, a nucleic acid polymer, a nucleotide analog, a nucleosideanalog, a nucleoside triphosphate, a deoxynucleoside triphosphate, adideoxynucleoside triphosphate, a hapten, a carbohydrate, apolysaccharide, a lipid, an ion-complexing moiety (such as a crownether), a PEG group and an organic or inorganic polymer. In certainembodiments, at least one member selected from R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, R¹⁰, R¹², R¹³, R¹⁹ and R²⁰ is a conjugated substance. In certainembodiments, at least one of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ isa conjugated substance. In certain embodiments, at least one of R⁷, R⁸,R⁹, R¹⁰, R¹² and R¹³ is a conjugated substance. In certain embodiments,at least one of R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴,R¹⁵ and R¹⁶ is a PEG group.

In certain embodiments, compounds are provided selected from the groupconsisting of Compound 1, Compound 2, Compound 3, Compound 4, Compound5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16,Compound 17, Compound 18, Compound 19, Compound 20, Compound 21,Compound 22, Compound 23, Compound 24, Compound 25 and amine-reactiveforms thereof.

In certain embodiments, compositions are provided, the compositionscomprising:

a) one or more of the compounds provided herein; and

b) a carrier.

In certain embodiments, compositions are provided, the compositionscomprising:

a) one or more of the compounds provided herein; and

b) an analyte.

In certain embodiments, compositions for tracking cell proliferation,differentiation and/or function are provided, the compositionscomprising:

a) one or more of the compounds provided herein; and

b) a carrier,

wherein the one or more of the compounds are present in an amounteffective to track cell proliferation, differentiation, and/or function.

In certain embodiments, compositions for tracking cell proliferation,differentiation and/or function are provided, the compositionscomprising:

(a) one or more of the compounds provided herein; and

(b) an analyte,

wherein the one or more of the compounds are present in an amounteffective to track cell proliferation, differentiation, and/or function.

In certain embodiments, the analyte is a cell and the compound islocated inside the cell. In certain embodiments, the compound isconjugated to a carrier molecule.

Another embodiment provides a kit for tracking cell proliferation,differentiation and/or function, the kit being compatible for use with,for example, flow cytometry and fluorescence microscopy, and comprising:

a) a compound of structural formula (I):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵, and R¹⁶ are eachindependently H, halogen, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c); or

R⁷ taken together with R¹⁴ are part of an optionally substituted 5- or6-membered ring;

R⁸ taken together with R¹⁵ are part of an optionally substituted 5- or6-membered ring;

R⁹ taken together with R¹¹ are part of an optionally substituted 5- or6-membered ring;

R¹⁰ taken together with R¹⁶ are part of an optionally substituted 5- or6-membered ring;

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) an organic solvent; and

c) a desiccant.

In certain embodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). Incertain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are each independently H,alkyl, substituted alkyl, or sulfoalkyl. In certain embodiments, R⁷, R⁸,R⁹, and R¹⁰ are each independently a C₁-C₆ alkyl, which can be the sameor different. In certain embodiments, R⁷, R¹, R⁹, and R¹⁰ are eachmethyl. In certain embodiments, R¹² and R¹³ are each alkyl, preferably aC₁-C₆ alkyl. In certain embodiments, R¹² and R¹³ are each methyl. Incertain embodiments, R¹¹ and R¹⁴ are each independently alkyl,substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certainembodiments, R¹¹ and R¹⁴ are each independently a C₁-C₆ alkyl, a C₁-C₆substituted alkyl, or sulfoalkyl, which can be the same or different. Incertain embodiments, R_(x) is succinimidyl ester (SE),sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP) ester,tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a kit for tracking cell proliferation,differentiation and/or function, the kit being compatible for use with,for example, flow cytometry and fluorescence microscopy, and comprising:

a) a compound of structural formula (II):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹ and R²²are each independently H, halogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) an organic solvent; and

c) a desiccant.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R³, R⁴, R⁵ and R⁶ are each independently H or halo. Incertain embodiments, R³, R⁴, R⁵ and R⁶ are independently H, Cl, or F. Incertain embodiments, R⁷ and R⁹ are each independently alkyl, substitutedalkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certain embodiments,R⁷ and R⁹ are each independently methyl, -L-R_(x), -L-S_(c) or(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 3. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a kit for tracking cell proliferation,differentiation and/or function, the kit being compatible for use with,for example, flow cytometry and fluorescence microscopy, and comprising:

a) a compound of structural formula (III):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³,and R²⁴ are each independently H, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) an organic solvent; and

c) a desiccant.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R⁷, R⁹, R¹⁷, R¹⁹, R²⁰, R²², R²³ and R²⁴ are eachindependently alkyl, substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or-L-S_(c). In certain embodiments, R⁷, R⁹, R¹⁷, R²², R²³ and R²⁴ are eachmethyl. In certain embodiments, R¹⁹ and R²⁰ are each independently(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 1. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a kit for tracking cell proliferation,differentiation and/or function, the kit being compatible for use with,for example, flow cytometry and fluorescence microscopy, and comprising:

a) a compound of structural formula (IV):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³,and R²⁴ are each independently H, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) an organic solvent; and

c) a desiccant.

In certain embodiments, R⁹, R¹², R¹³, R²² and R²³ are each alkyl,preferably a C₁-C₆ alkyl. In certain embodiments, R⁹, R¹², R¹³, R²² andR²³ are each methyl. In certain embodiments, R³, R⁴, R⁵ and R⁶ are eachindependently H or halo. In certain embodiments, R³ and R⁶ areindependently H, Cl, or F. In certain embodiments, R² is carboxyl. Incertain embodiments, R⁷, R⁹ and R²⁰ are each independently alkyl,substituted alkyl, sulfoalkyl, sulfo, (CH₂)_(n)SO₃ ⁻, wherein n is aninteger between 1 and 6, -L-R_(x), or -L-S_(c). In certain embodiments,R⁷ is -L-R_(x) and R²⁰ is (CH₂)_(n)SO₃ ⁻. In certain embodiments, nis 1. In certain embodiments, R_(x) is succinimidyl ester (SE),sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP) ester,tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

In certain embodiments, R_(x) is selected from an acrylamide, anactivated ester of a carboxylic acid, a carboxylic ester, an acyl azide,an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline,an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a photoactivatable group, a reactive platinum complex,a silyl halide, a sulfonyl halide, and a thiol. In certain embodimentsthe reactive group is selected from the group consisting of carboxylicacid, succinimidyl ester of a carboxylic acid, hydrazide, amine and amaleimide. The reactive group may be attached to any appropriate site onthe reporter molecule or the aniline moiety. In certain embodiments, atleast one member selected from R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹²,R¹³, R¹⁹ and R²⁰ is a reactive group. In certain embodiments, at leastone of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is a reactive group. Incertain embodiments, at least one of R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is areactive group.

In certain embodiments, S_(c) is selected from a carrier molecule and asolid support. In certain embodiments, S_(c) is selected from an aminoacid, a polymer of amino acids, a peptide, a protein, a neurotoxin, aphallotoxin, a cytokine, a toxin, a protease substrate, a protein kinasesubstrate, an enzyme, an antibody, an antibody fragment, a lectin, aglycoprotein, a histone, an albumin, a lipoprotein, avidin,streptavidin, protein A, protein G, a phycobiliprotein, a fluorescentprotein, a hormone, a growth factor, a nucleic acid base, a nucleoside,a nucleotide, a nucleic acid polymer, a nucleotide analog, a nucleosideanalog, a nucleoside triphosphate, a deoxynucleoside triphosphate, adideoxynucleoside triphosphate, a hapten, a carbohydrate, apolysaccharide, a lipid, an ion-complexing moiety (such as a crownether), a PEG group, and an organic or inorganic polymer. In certainembodiments, at least one member selected from R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹² and R¹³ is a conjugated substance. In certainembodiments, at least one of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁹and R²⁰ is a conjugated substance. In certain embodiments, at least oneof R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is a conjugated substance. In certainembodiments, at least one of R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹,R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ is a PEG group.

In certain embodiments, the compounds used in the kits provided hereinare selected from the group consisting of Compound 1, Compound 2,Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8,Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19,Compound 20, Compound 21, Compound 22, Compound 23, Compound 24,Compound 25 and amine-reactive forms thereof.

In another illustrative embodiment of the kit, R_(X) is a succinimidylester. In another illustrative embodiment, the organic solvent is DMSO.In another illustrative embodiment, the kit further comprisesinstructions for tracking cell proliferation, differentiation, and/orfunction according to a method disclosed herein.

In certain embodiments, kits for tracking cell proliferation,differentiation and/or function are provided, the kit comprising:

(a) one or more of the compounds described herein;

(b) one or more containers; and optionally

(c) instructions for tracking cell proliferation, differentiation,and/or function according to a method disclosed herein.

In certain embodiments, kits are provided, the kit comprising:

(a) one or more of the compounds described herein;

(b) one or more containers; and optionally

(c) instructions for using according to a method disclosed herein.

In certain embodiments, kits for tracking cell proliferation,differentiation and/or function are provided, the kit comprising:

(a) one or more of the compositions described herein;

b) an organic solvent; and

c) a desiccant.

In certain embodiments, kits for tracking cell proliferation,differentiation and/or function are provided, the kit comprising:

(a) one or more of the compositions described herein;

(b) one or more containers; and optionally

(c) instructions for tracking cell proliferation, differentiation,and/or function according to a method disclosed herein.

Another embodiment provides a method for tracking cell proliferation,differentiation, and/or function, the method being compatible for usewith, for example, flow cytometry and fluorescence microscopy, andcomprising:

a) incubating a mixture of cells and a compound of structural formula(I):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵, and R¹⁶ are eachindependently H, halogen, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c); or

R⁷ taken together with R¹⁴ are part of an optionally substituted 5- or6-membered ring;

R⁸ taken together with R¹⁵ are part of an optionally substituted 5- or6-membered ring;

R⁹ taken together with R¹¹ are part of an optionally substituted 5- or6-membered ring;

R¹⁰ taken together with R¹⁶ are part of an optionally substituted 5- or6-membered ring;

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) providing a stimulus to the mixture to elicit a fluorescent signal;and

c) analyzing the stimulated mixture.

In certain embodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). Incertain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are each independently H,alkyl, substituted alkyl, or sulfoalkyl. In certain embodiments, R⁷, R⁸,R⁹, and R¹⁰ are each independently a C₁-C₆ alkyl, which can be the sameor different. In certain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are eachmethyl. In certain embodiments, R¹² and R¹³ are each alkyl, preferably aC₁-C₆ alkyl. In certain embodiments, R¹² and R¹³ are each methyl. Incertain embodiments, R¹¹ and R¹⁴ are each independently alkyl,substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certainembodiments, R¹¹ and R¹⁴ are each independently a C₁-C₆ alkyl, a C₁-C₆substituted alkyl, or sulfoalkyl, which can be the same or different. Incertain embodiments, R_(x) is succinimidyl ester (SE),sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP) ester,tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a method for tracking cell proliferation,differentiation, and/or function, the method being compatible for usewith, for example, flow cytometry and fluorescence microscopy, andcomprising:

a) incubating a mixture of cells and a compound of structural formula(II):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl,-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹ and R²²are each independently H, halogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) providing a stimulus to the mixture to elicit a fluorescent signal;and

c) analyzing the stimulated mixture.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R³, R⁴, R⁵ and R⁶ are each independently H or halo. Incertain embodiments, R³, R⁴, R⁵ and R⁶ are independently H, Cl, or F. Incertain embodiments, R⁷ and R⁹ are each independently alkyl, substitutedalkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certain embodiments,R⁷ and R⁹ are each independently methyl, -L-R_(x), -L-S_(c) or(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 3. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a method for tracking cell proliferation,differentiation, and/or function, the method being compatible for usewith, for example, flow cytometry and fluorescence microscopy, andcomprising:

a) incubating a mixture of cells and a compound of structural formula(III):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³,and R²⁴ are each independently H, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) providing a stimulus to the mixture to elicit a fluorescent signal;and

c) analyzing the stimulated mixture.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R⁷, R⁹, R¹⁷, R¹⁹, R²⁰, R²², R²³ and R²⁴ are eachindependently alkyl, substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or-L-S_(c). In certain embodiments, R⁷, R⁹, R¹⁷, R²², R²³ and R²⁴ are eachmethyl. In certain embodiments, R¹⁹ and R²⁰ are each independently(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 1. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a method for tracking cell proliferation,differentiation, and/or function, the method being compatible for usewith, for example, flow cytometry and fluorescence microscopy, andcomprising:

a) incubating a mixture of cells and a compound of structural formula(IV):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³,and R²⁴ are each independently H, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) providing a stimulus to the mixture to elicit a fluorescent signal;and

c) analyzing the stimulated mixture.

In certain embodiments, R⁹, R¹², R¹³, R²² and R²³ are each alkyl,preferably a C₁-C₆ alkyl.

In certain embodiments, R⁹, R¹², R¹³, R²² and R²³ are each methyl. Incertain embodiments, R³, R⁴, R⁵ and R⁶ are each independently H or halo.In certain embodiments, R³ and R⁶ are independently H, Cl, or F. Incertain embodiments, R² is carboxyl. In certain embodiments, R⁷, R⁹ andR²⁰ are each independently alkyl, substituted alkyl, sulfoalkyl, sulfo,(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6, -L-R_(x), or-L-S_(c). In certain embodiments, R⁷ is -L-R_(x) and R²⁰ is (CH₂)_(n)SO₃⁻. In certain embodiments, n is 1. In certain embodiments, R_(x) issuccinimidyl ester (SE), sulfodichlorophenyl (SDP) ester,sulfotetrafluorophenyl (STP) ester, tetrafluorophenyl (TFP) ester,pentafluorophenyl (PFP) ester, nitrilotriacetic acid (NTA),aminodextran, and cyclooctyne-amine.

In certain embodiments, R_(x) is selected from an acrylamide, anactivated ester of a carboxylic acid, a carboxylic ester, an acyl azide,an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline,an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a photoactivatable group, a reactive platinum complex,a silyl halide, a sulfonyl halide, and a thiol. In certain embodimentsthe reactive group is selected from the group consisting of carboxylicacid, succinimidyl ester of a carboxylic acid, hydrazide, amine and amaleimide. The reactive group may be attached to any appropriate site onthe reporter molecule or the aniline moiety. In certain embodiments, atleast one member selected from R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹²,R¹³, R¹⁹ and R²⁰ is a reactive group. In certain embodiments, at leastone of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is a reactive group. Incertain embodiments, at least one of R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is areactive group.

In certain embodiments, S_(c) is selected from a carrier molecule and asolid support. In certain embodiments, S_(c) is selected from an aminoacid, a polymer of amino acids, a peptide, a protein, a neurotoxin, aphallotoxin, a cytokine, a toxin, a protease substrate, a protein kinasesubstrate, an enzyme, an antibody, an antibody fragment, a lectin, aglycoprotein, a histone, an albumin, a lipoprotein, avidin,streptavidin, protein A, protein G, a phycobiliprotein, a fluorescentprotein, a hormone, a growth factor, a nucleic acid base, a nucleoside,a nucleotide, a nucleic acid polymer, a nucleotide analog, a nucleosideanalog, a nucleoside triphosphate, a deoxynucleoside triphosphate, adideoxynucleoside triphosphate, a hapten, a carbohydrate, apolysaccharide, a lipid, an ion-complexing moiety (such as a crownether), a PEG group, and an organic or inorganic polymer. In certainembodiments, at least one member selected from R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁹ and R²⁰ is a conjugated substance. In certainembodiments, at least one of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ isa conjugated substance. In certain embodiments, at least one of R⁷, R⁸,R⁹, R¹⁰, R¹² and R¹³ is a conjugated substance. In certain embodiments,at least one of R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴,R¹⁵ and R¹⁶ is a PEG group.

In certain embodiments, the compounds used in the methods providedherein are selected from the group consisting of Compound 1, Compound 2,Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8,Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19,Compound 20, Compound 21, Compound 22, Compound 23, Compound 24,Compound 25 and amine-reactive forms thereof.

In one illustrative embodiment, the method includes a second compoundexcitable at a different wavelength as the first compound. In anotherillustrative embodiment, the method includes a second compound where thesecond compound includes, for example, GFP or PACIFIC BLUE (Termo FisherScientific).

In another illustrative embodiment of the method, step a) is conductedfor approximately 20 minutes. In another illustrative embodiment, stepb) and step c) are carried out concurrently. In another illustrativeembodiment, step b) and step c) involve flow cytometry.

The present disclosure also provides methods for determining cell healthand/or viability using one or more of the compounds provided herein.

The present disclosure also provides methods for detecting an analyte ina sample using one or more of the compounds provided herein.

Another illustrative embodiment provides a process for preparing aconjugated compound of structural formula (I), (II), (III) or (IV), theprocess comprising:

reacting compound of structural formula (I), (II), (III) or (IV) with asubstance to be conjugated thereto, thereby resulting in a conjugatedsubstance S_(c).

In certain embodiments, the compound of structural formula (I), (II),(III) or (IV) is conjugated to a carrier molecule or solid support. Incertain embodiments, the compound of structural formula (I), (II), (III)or (IV) is conjugated to molecule selected from an amino acid, a polymerof amino acids, a peptide, a protein, a neurotoxin, a phallotoxin, acytokine, a toxin, a protease substrate, a protein kinase substrate, anenzyme, an antibody, an antibody fragment, a lectin, a glycoprotein, ahistone, an albumin, a lipoprotein, avidin, streptavidin, protein A,protein G, a phycobiliprotein, a fluorescent protein, a hormone, agrowth factor, a nucleic acid base, a nucleoside, a nucleotide, anucleic acid polymer, a nucleotide analog, a nucleoside analog, anucleoside triphosphate, a deoxynucleoside triphosphate, adideoxynucleoside triphosphate, a hapten, a carbohydrate, apolysaccharide, a lipid, an ion-complexing moiety (such as a crownether), a PEG group, and an organic or inorganic polymer.

Other illustrative aspects, features and advantages of the presentdisclosure will become apparent from the following detailed description.It should be understood, however, that the detailed description and thespecific examples that follow, while indicating preferred embodimentsare given by way of illustration only. It is expected that variouschanges and modifications within the spirit and scope of the presentdisclosure will become apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B: Comparison of cell tracing ability of Compound 2 (FIG.1A) and CELLTRACE Far Red DDAO (FIG. 1B), demonstrating that Compound 2is able to trace seven generations of cells (see, Example 2).

FIG. 2A: Dual parameter plot of PBMCs using SYTOX Green (Thermo FisherScientific) to gate for live cells for proliferation analysis withCompound 12 (see, Example 2).

FIG. 2B: Histogram showing generational peaks of PBMCs using Compound12.

FIG. 3A: Dual parameter plot of PBMCs using SYTOX Green to gate for livecells for proliferation analysis with Compound 3 (see, Example 2).

FIG. 3B: Histogram showing generational peaks of PBMCs using Compound 3.

FIG. 4: Cell imaging analysis showing cell permeability of Compound 2.

FIG. 5: Wound healing assay using Compound 2 (see, Example 3).

FIG. 6: Angiogenesis assay using Compound 2 (see, Example 4).

FIG. 7: Differentiation assay using Compound 2 (see, Example 5).

FIG. 8: Bioconjugation results of reacting goat-anti-mouse (GAM) IgGwith Compound 18 (see, Example 6).

FIG. 9A: Flow cytometry histograms showing signal from lymphocytesstained with APC-CD4 conjugate in four emission channels: BL1-H (530/30nm), BL2-H (574/26 nm), BL3 (690/50 nm), BL4 (780/60), RL1 (660/20 nm),RL2-H (780/60 nm) (see, Example 8).

FIG. 9B: Flow cytometry histograms showing signal from lymphocytesstained with Compound 18-CD4 conjugate in four emission channels: BL1-H(530/30 nm), BL2-H (574/26 nm), BL3 (690/50 nm), BL4 (780/60 nm), RL1(660/20 nm), RL2-H (780/60 nm). Note the lack of spillover in BL3-H,BL4-H, and RL1-H, compared with that from APC-CD4 in FIG. 9A; Compound18-CD4 enables bright signal specifically in RL2, in contrast to signalin multiple channels using APC-CD4 (see, Example 8).

FIGS. 9C and 9D: Flow cytometry histograms showing signal in the RL2-Hchannel (780/60 nm) from lymphocytes stained with tandem dye conjugatesAPC-ALEXA FLUOR 750-CD4 (left) and APC-Compound 18-CD4 (right). (see,Example 8) FIG. 9E: Flow cytometry histograms showing signal fromlymphocytes stained with tandem dye conjugates of CD4 in the RL2-Hchannel (705/40 nm). Left histogram shows signal from cells stained withAPC-ALEXA FLUOR 750-CD4, middle histogram shows signal from APC-Compound18-CD4 DOL 5, and right histogram shows signal from APC-Compound 18-CD4DOL10. The term DOL refers to the number of molecules of Compound 18 pernumber of molecules of APC in the tandem conjugates. The Table shows theimproved SI (Staining Index) and Signal/Noise (S:N) of conjugates madewith Compound 18 over those made with ALEXA FLUOR 750 (Thermo FisherScientific) (see, Example 8).

FIG. 10: Lack of cytotoxicity of Compound 2, as measured by the CYQUANTDirect (Thermo Fisher Scientific) method. Fluorescence on the Y-axis isproportional to cell population viability. A549 cells plated in a 96well plate were labeled with CELLTRACKER Blue, Green, Red, Compound 2,or DMSO in Live Cell Imaging Solution at 10 μM concentration for 30 minsat 37° C. The LCIS was then aspirated from cells and complete mediaadded for an overnight incubation. The following day (Day 1) cells wereassayed for cytotoxicity using the CYQUANT Direct assay (Thermo FisherScientific). The CYQUANT Direct assay was repeated on cells from thesame 96 well plate on days 2 and 3 showing no cytotoxicity ofCELLTRACKER Blue, Green, Red or Compound 2 when compared to DMSO alone(see, Example 9).

DETAILED DESCRIPTION

The present disclosure provides compounds, methods, and kits for short-and long-term tracking of cell proliferation, differentiation, structureand/or function, as well as for staining samples, labeling proteins,detecting ligands and other analytes. The compounds of the presentdisclosure are dye compounds that fluoresce in the red portion of theUV/VIS spectrum and provide bright fluorescence intensity, uniform cellstaining, good retention within cells and have low toxicity towardcells. The compounds disclosed herein are carbopyronine-based dyecompounds, including reactive dye compound derivatives anddye-conjugates. These compounds may be used in place of and/or incombination with other currently-available cell analysis reagents, suchas, for example, the 488 nm-excitable reagents IgG antibodies conjugatedwith fluorescein (FAM) and/or Green Fluorescent Protein (GFP) to trackand/or stain otherwise indistinguishable cell populations in mixed cellcultures via flow cytometry and/or fluorescence microscopy,respectively. The present disclosure also includes processes forpreparing and using the compounds described herein in the disclosedmethods and kits provided herein.

Definitions

To more clearly and concisely describe and point out the subject matterof the present disclosure, the following definitions are provided forspecific terms, which are used in the following description and appendedclaims. Throughout the specification, exemplification of specific termsshould be considered as non-limiting examples.

Before describing the present teachings in detail, it is to beunderstood that the disclosure is not limited to specific compositionsor process steps, as such may vary. It should be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. Thus, for example, reference to “a compound” includes aplurality of compounds and reference to “a cell” includes a plurality ofcells and the like. The phrase “and/or” denotes a shorthand way ofindicating that the specific combination is contemplated in combinationand separately, in the alternative. For illustration purposes, but notas a limitation, “X and/or Y” can mean “X” or “Y” or “X” and “Y”.

It will be appreciated that there is an implied “about” prior to thetemperatures, concentrations, times, etc. discussed in the presentdisclosure, such that slight and insubstantial deviations are within thescope of the present teachings herein. Also, the use of “comprise”,“comprises”, “comprising”, “contain”, “contains”, “containing”,“include”, “includes”, and “including” are not intended to be limiting.It is to be understood that both the foregoing general description anddetailed description are exemplary and explanatory only and are notrestrictive of the teachings.

Unless specifically noted in the above specification, embodiments in thespecification that recite “comprising” various components are alsocontemplated as “consisting of” or “consisting essentially of” therecited components; embodiments in the specification that recite“consisting of” various components are also contemplated as “comprising”or “consisting essentially of” the recited components; and embodimentsin the specification that recite “consisting essentially of” variouscomponents are also contemplated as “consisting of” or “comprising” therecited components (this interchangeability does not apply to the use ofthese terms in the claims).

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed terms preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the desired subject matter inany way. All literature cited in the specification, including but notlimited to, patents, patent applications, articles, books and treatisesare expressly incorporated by reference in their entirety for anypurpose. In the event that any of the incorporated literaturecontradicts any term defined in this specification, this specificationcontrols. While the present teachings are described in conjunction withvarious embodiments, it is not intended that the present teachings belimited to such embodiments. On the contrary, the present teachingsencompass various alternatives, modifications, and equivalents, as willbe appreciated by those of skill in the art.

A dashed line projecting from a substituent, such as:

indicates the point of attachment to the base molecule. For a fusedring, dashed lines indicate portions of the base molecule where thefused ring is attached, such as:

wherein the full molecule could have the structure:

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

It is understood that in all substituted groups defined herein, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups with two other substituted aryl groups are limited to-substituted aryl-(substituted aryl)-substituted aryl.

Similarly, it is understood that the definitions provided herein are notintended to include impermissible substitution patterns (e.g., methylsubstituted with 5 fluoro groups). Such impermissible substitutionpatterns are well known to the skilled artisan.

The compounds disclosed herein may exist in unsolvated forms as well assolvated forms, including hydrated forms. These compounds may exist inmultiple crystalline or amorphous forms. In general, all physical formsare equivalent for the uses described herein and are intended to bewithin the scope of the present disclosure. The compounds disclosedherein may possess asymmetric carbon atoms (i.e., chiral centers) ordouble bonds; the racemates, diastereomers, geometric isomers andindividual isomers of the compounds described herein are within thescope of the present disclosure. The compounds described herein may beprepared as a single isomer or as a mixture of isomers.

Where substituent groups are specified by their conventional chemicalformulae and are written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

It will be understood that the chemical structures that are used todefine the compounds disclosed herein are each representations of one ofthe possible resonance structures by which each given structure can berepresented. Further, it will be understood that by definition,resonance structures are merely a graphical representation used by thoseof skill in the art to represent electron delocalization, and that thepresent disclosure is not limited in any way by showing one particularresonance structure for any given structure.

Where a disclosed compound includes a conjugated ring system, resonancestabilization may permit a formal electronic charge to be distributedover the entire molecule. While a particular charge may be depicted aslocalized on a particular ring system, or a particular heteroatom, it iscommonly understood that a comparable resonance structure can be drawnin which the charge may be formally localized on an alternative portionof the compound.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupshaving from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms,e.g. 1, 2, 3, 4, 5 or 6 carbon atoms. This term includes, by way ofexample, linear and branched hydrocarbyl groups such as methyl (CH₃—),ethyl (CH₃CH₂—), n-propyl (CH₃CH₂CH₂—), isopropyl ((CH₃)₂CH—), n-butyl(CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—), sec-butyl ((CH₃)(CH₃CH₂)CH—),t-butyl ((CH₃)₃C—), n-pentyl (CH₃CH₂CH₂CH₂CH₂—), and neopentyl((CH₃)₃CCH₂—).

“Substituted alkyl” refers to an alkyl group having from 1 to 5,preferably 1 to 3, or more preferably 1 to 2 substituents selected fromthe group consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,substituted aryl, aryloxy, substituted aryloxy, arylthio, substitutedarylthio, carboxyl, carboxylalkyl, carboxyl ester, (carboxylester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substitutedcycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio,substituted cycloalkylthio, cycloalkenyl, substituted cycloalkenyl,cycloalkenyloxy, substituted cycloalkenyloxy, cycloalkenylthio,substituted cycloalkenylthio, guanidino, substituted guanidino, halo,hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substitutedheteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclic,substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy,heterocyclylthio, substituted heterocyclylthio, nitro, sulfo,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, andsubstituted alkylthio, wherein said substituents are defined herein.Particular substituted alkyl groups comprise a reactive group for director indirect linking to a carrier molecule or solid support, for example,but not limited to, alkyl substituted by carboxyl or a carboxyl ester(e.g. an activated ester such as an N-hydroxysuccinimide ester) andalkyl substituted by aminocarbonyl —CONHR where R is an organic moietyas defined below with reference to the term “aminocarbonyl”, e.g. aC₁-C₁₀ (e.g. C₁-C₆) alkyl terminally substituted by a reactive group(R_(x)) including, but not limited to, carboxyl, carboxylester,maleimide, succinimidyl ester (SE), sulfodichlorophenyl (SDP) ester,sulfotetrafluorophenyl (STP) ester, tetrafluorophenyl (TFP) ester,pentafluorophenyl (PFP) ester, nitrilotriacetic acid (NTA),aminodextran, and cyclooctyne-amine.

“Alkoxy” refers to the group —O-alkyl wherein alkyl is defined herein.Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and n-pentoxy.

“Substituted alkoxy” refers to the group —O-(substituted alkyl), whereinsubstituted alkyl is defined herein.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl) which condensed rings may ormay not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is at an aromatic carbon atom. Preferred aryl groupsinclude phenyl and naphthyl.

“Substituted aryl” refers to aryl groups which are substituted with 1 to5, preferably 1 to 3, or more preferably 1 to 2 substituents selectedfrom the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl,aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substitutedcycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy,cycloalkenylthio, substituted cycloalkenylthio, guanidino, substitutedguanidino, halo, hydroxy, heteroaryl, substituted heteroaryl,heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substitutedheteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy,substituted heterocyclyloxy, heterocyclylthio, substitutedheterocyclylthio, nitro, sulfo, substituted sulfonyl, sulfonyloxy,thioacyl, thiol, alkylthio, and substituted alkylthio, wherein saidsubstituents are defined herein.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen and sulfur within the ring. Such heteroaryl groups can have asingle ring (e.g., pyridinyl or furyl) or multiple condensed rings(e.g., indolizinyl or benzothienyl) wherein the condensed rings may ormay not be aromatic and/or contain a heteroatom provided that the pointof attachment is through an atom of the aromatic heteroaryl group. Inone embodiment, the nitrogen and/or the sulfur ring atom(s) of theheteroaryl group are optionally oxidized to provide for the N-oxide(N→O), sulfinyl, or sulfonyl moieties. Preferred heteroaryls includepyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.

“Substituted heteroaryl” refers to heteroaryl groups that aresubstituted with from 1 to 5, preferably 1 to 3, or more preferably 1 to2 substituents selected from the group consisting of the same group ofsubstituents defined for substituted aryl.

“Heteroaryloxy” refers to —O-heteroaryl.

“Substituted heteroaryloxy” refers to the group —O-(substitutedheteroaryl).

“Alkenyl” refers to alkenyl groups having from 2 to 6 carbon atoms andpreferably 2 to 4 carbon atoms and having at least 1 and preferably from1 to 2 sites of alkenyl unsaturation. Such groups are exemplified, forexample, by vinyl, allyl, but-3-en-1-yl, and propenyl.

“Substituted alkenyl” refers to alkenyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,substituted aryl, aryloxy, substituted aryloxy, arylthio, substitutedarylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy,substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substitutedcycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substitutedheteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio,substituted heteroarylthio, heterocyclic, substituted heterocyclic,heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,substituted heterocyclylthio, nitro, sulfo, substituted sulfonyl,sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,wherein said substituents are defined herein and with the proviso thatany hydroxy substitution is not attached to a vinyl (unsaturated) carbonatom.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O)—, heterocyclic-C(O)—, and substitutedheterocyclic-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein. Acyl includes the“acetyl” group CH₃C(O)—.

“Acylamino” refers to the groups —NRC(O)alkyl, —NRC(O) substitutedalkyl, —NRC(O) cycloalkyl, —NRC(O) substituted cycloalkyl,—NRC(O)cycloalkenyl, —NRC(O) substituted cycloalkenyl, —NRC(O)alkenyl,—NRC(O) substituted alkenyl, —NRC(O)alkynyl, —NRC(O) substitutedalkynyl, —NRC(O)aryl, —NRC(O) substituted aryl, —NRC(O)heteroaryl,—NRC(O) substituted heteroaryl, —NRC(O)heterocyclic, and —NRC(O)substituted heterocyclic, wherein R is hydrogen or alkyl and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Acyloxy” refers to the groups alkyl-C(O)O—, substituted alkyl-C(O)O—,alkenyl-C(O)O—, substituted alkenyl-C(O)O—, alkynyl-C(O)O—, substitutedalkynyl-C(O)O—, aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—,substituted cycloalkyl-C(O)O—, cycloalkenyl-C(O)O—, substitutedcycloalkenyl-C(O)O—, heteroaryl-C(O)O—, substituted heteroaryl-C(O)O—,heterocyclic-C(O)O—, and substituted heterocyclic-C(O)O—, wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

“Amino” refers to the group —NH₂.

“Substituted amino” refers to the group —NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl,—SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-cycloalkenyl,—SO₂-substituted cylcoalkenyl, —SO₂-aryl, —SO₂-substituted aryl,—SO₂-heteroaryl, —SO₂-substituted heteroaryl, —SO₂-heterocyclic, and—SO₂-substituted heterocyclic and wherein R′ and R″ are optionallyjoined, together with the nitrogen bound thereto to form a heterocyclicor substituted heterocyclic group, provided that R′ and R″ are both nothydrogen, and wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein. When R′ is hydrogen and R″ is alkyl,the substituted amino group is sometimes referred to herein asalkylamino. When R′ and R″ are alkyl, the substituted amino group issometimes referred to herein as dialkylamino. When referring to amonosubstituted amino, it is meant that either R′ or R″ is hydrogen butnot both. When referring to a disubstituted amino, it is meant thatneither R′ nor R″ are hydrogen.

“Aminocarbonyl” refers to the group —C(O)NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic, and where R′ andR″ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Aminothiocarbonyl” refers to the group —C(S)NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic, and where R′ andR″ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Aminocarbonylamino” refers to the group —NRC(O)NR′R″ where R ishydrogen or alkyl and R′ and R″ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic, and where R′ and R″ are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Aminothiocarbonylamino” refers to the group —NRC(S)NR′R″ where R ishydrogen or alkyl and R′ and R″ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic, and where R′ and R″ are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Aminocarbonyloxy” refers to the group —O—C(O)NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R′ andR″ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Aminosulfonyl” refers to the group —SO₂NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic, and where R′ andR″ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Aminosulfonyloxy” refers to the group —O—SO₂NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic, and where R′ andR″ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Aminosulfonylamino” refers to the group —NRSO₂NR′R″ where R″ ishydrogen or alkyl and R′ and R″ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkyenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic, and where R′ and R″ are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkyenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Amidino” refers to the group —C(═NR′″)R′R″ where R′, R″, and R′″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic, and where R′ andR″ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic are asdefined herein.

“Aryloxy” refers to the group —O-aryl, where aryl is as defined herein,that includes, by way of example, phenoxy and naphthoxy.

“Substituted aryloxy” refers to the group —O-(substituted aryl), wheresubstituted aryl is as defined herein.

“Arylthio” refers to the group —S-aryl, where aryl is as defined herein.

“Substituted arylthio” refers to the group —S-(substituted aryl), wheresubstituted aryl is as defined herein.

“Alkynyl” refers to alkynyl groups having from 2 to 6 carbon atoms andpreferably 2 to 3 carbon atoms and having at least 1 and preferably from1 to 2 sites of alkynyl unsaturation.

“Substituted alkynyl” refers to alkynyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,substituted aryl, aryloxy, substituted aryloxy, arylthio, substitutedarylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy,substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substitutedcycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substitutedheteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio,substituted heteroarylthio, heterocyclic, substituted heterocyclic,heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,substituted heterocyclylthio, nitro, sulfo, substituted sulfonyl,sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,wherein said substituents are defined herein and with the proviso thatany hydroxy substitution is not attached to an acetylenic carbon atom.

“Carbonyl” refers to the divalent group —C(O)— which is equivalent to—C(═O)—.

“Carboxyl” or “carboxy” refers to —COOH or salts thereof.

“Carboxyl alkyl” or “carboxyalkyl” refers to the group —(CH₂)_(n)COOH,where n is 1-6.

“Carboxyl ester” or “carboxy ester” refers to the groups —C(O)O-alkyl,—C(O)O— substituted alkyl, —C(O)O-alkenyl, —C(O)O-substituted alkenyl,—C(O)O-alkynyl, —C(O)O— substituted alkynyl, —C(O)O-aryl,—C(O)O-substituted aryl, —C(O)O-cycloakyl, —C(O)O— substitutedcycloalkyl, —C(O)O-cycloalkenyl, —C(O)O-substituted cycloalkenyl,—C(O)O— heteroaryl, —C(O)O-substituted heteroaryl, —C(O)O-heterocyclic,and —C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“(Carboxyl ester)amino” refers to the group —NR—C(O)O-alkyl, —NR)C(O)O—substituted alkyl, —NR—C(O)O-alkenyl, —NR—C(O)O-substituted alkenyl,—NR—C(O)O-alkynyl, —NR—C(O)O— substituted alkynyl, —NR—C(O)O-aryl,—NR—C(O)O-substituted aryl, —NR—C(O)O-cycloalkyl, —NR—C(O)O-substitutedcycloalkyl, —NR—C(O)O-cycloalkenyl, —NR—C(O)O-substituted cycloalkenyl,—NR—C(O)O-heteroaryl, —NR—C(O)O-substituted heteroaryl,—NR—C(O)O-heterocyclic, and —NR—C(O)O-substituted heterocyclic, whereinR is alkyl or hydrogen, and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“(Carboxyl ester)oxy” refers to the group —O—C(O)O-alkyl, —O—C(O)O—substituted alkyl, —O—C(O)O-alkenyl, —O—C(O)O-substituted alkenyl,—O—C(O)O-alkynyl, —O—C(O)O-substituted alkynyl, —O—C(O)O-aryl,—O—C(O)O-substituted aryl, —O—C(O)O-cycloalkyl, —O—C(O)O-substitutedcycloalkyl, —O—C(O)O-cycloalkenyl, —O—C(O)O-substituted cycloalkenyl,—O—C(O)O— heteroaryl, —O—C(O)O-substituted heteroaryl,—O—C(O)O-heterocyclic, and —O—C(O)O-substituted heterocyclic, whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Cyano” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andspiro ring systems. Examples of suitable cycloalkyl groups include, forinstance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, andcyclooctyl.

“Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to10 carbon atoms having single or multiple cyclic rings and having atleast one >C═C<ring unsaturation and preferably from 1 to 2 sitesof >C═C<ring unsaturation.

“Substituted cycloalkyl” and “substituted cycloalkenyl” refer to acycloalkyl or cycloalkenyl group having from 1 to 5 or preferably 1 to 3substituents selected from the group consisting of oxo, thione, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino,substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl,aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substitutedcycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy,cycloalkenylthio, substituted cycloalkenylthio, guanidino, substitutedguanidino, halo, hydroxy, heteroaryl, substituted heteroaryl,heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substitutedheteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy,substituted heterocyclyloxy, heterocyclylthio, substitutedheterocyclylthio, nitro, sulfo, substituted sulfonyl, sulfonyloxy,thioacyl, thiol, alkylthio, and substituted alkylthio, wherein saidsubstituents are defined herein.

“Cycloalkyloxy” refers to —O-cycloalkyl.

“Substituted cycloalkyloxy” refers to —O-(substituted cycloalkyl).

“Cycloalkylthio” refers to —S-cycloalkyl.

“Substituted cycloalkylthio” refers to —S-(substituted cycloalkyl).

“Cycloalkenyloxy” refers to —O-cycloalkenyl.

“Substituted cycloalkenyloxy” refers to —O-(substituted cycloalkenyl).

“Cycloalkenylthio” refers to —S-cycloalkenyl.

“Substituted cycloalkenylthio” refers to —S-(substituted cycloalkenyl).

“Guanidino” refers to the group —NHC(═NH)NH₂.

“Substituted guanidino” refers to —NR¹³C(═NR¹³)N(R¹³)₂ where each R¹³ isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and two R¹³groups attached to a common guanidino nitrogen atom are optionallyjoined together with the nitrogen bound thereto to form a heterocyclicor substituted heterocyclic group, provided that at least one R¹³ is nothydrogen, and wherein said substituents are as defined herein.

“H” indicates hydrogen.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen and sulfur within the ring. Such heteroaryl groups can have asingle ring (e.g., pyridinyl or furyl) or multiple condensed rings(e.g., indolizinyl or benzothienyl) wherein the condensed rings may ormay not be aromatic and/or contain a heteroatom provided that the pointof attachment is through an atom of the aromatic heteroaryl group. Inone embodiment, the nitrogen and/or the sulfur ring atom(s) of theheteroaryl group are optionally oxidized to provide for the N-oxide(N→O), sulfinyl, or sulfonyl moieties. Preferred heteroaryls includepyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.

“Substituted heteroaryl” refers to heteroaryl groups that aresubstituted with from 1 to 5, preferably 1 to 3, or more preferably 1 to2 substituents selected from the group consisting of the same group ofsubstituents defined for substituted aryl.

“Heteroaryloxy” refers to —O-heteroaryl.

“Substituted heteroaryloxy” refers to the group —O-(substitutedheteroaryl).

“Heteroarylthio” refers to the group —S-heteroaryl.

“Substituted heteroarylthio” refers to the group —S-(substitutedheteroaryl).

“Heterocycle” or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl”refers to a saturated or unsaturated group having a single ring ormultiple condensed rings, including fused bridged and spiro ringsystems, from 1 to 10 carbon atoms and from 1 to 4 hetero atoms selectedfrom the group consisting of nitrogen, sulfur or oxygen within the ringwherein, in fused ring systems, one or more the rings can be cycloalkyl,aryl or heteroaryl provided that the point of attachment is through thenon-aromatic ring. In one embodiment, the nitrogen and/or sulfur atom(s)of the heterocyclic group are optionally oxidized to provide for theN-oxide, sulfinyl, sulfonyl moieties.

“Substituted heterocyclic” or “substituted heterocycloalkyl” or“substituted heterocyclyl” refers to heterocyclyl groups that aresubstituted with from 1 to 5, or preferably 1 to 3 of the samesubstituents as defined for substituted cycloalkyl.

Examples of heterocycle and heteroaryls include, but are not limited to,azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, dihydroindole, indazole,purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,and tetrahydrofuranyl.

“Heterocyclyloxy” refers to the group —O-heterocyclyl.

“Substituted heterocyclyloxy” refers to the group —O-(substitutedheterocyclyl).

“Heterocyclylthio” refers to the group —S— heterocyclyl.

“Substituted heterocyclylthio” refers to the group —S-(substitutedheterocyclyl).

Examples of heterocycle and heteroaryls include, but are not limited to,azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, dihydroindole, indazole,purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,and tetrahydrofuranyl.

“Hydrazinyl” refers to the group —NHNH₂— or ═NNH—.

“Substituted hydrazinyl” refers to a hydrazinyl group, wherein anon-hydrogen atom, such as an alkyl group, is appended to one or both ofthe hydrazinyl amine groups. An example of substituted hydrazinyl is—N(alkyl)-NH₂ or ═N⁺(alkyl)-NH₂.

“Nitro” refers to the group —NO₂.

“Oxo” refers to the atom (═O) or (—O⁻).

“PEG groups” refer to ethylene glycol, diethylene glycol andpolyethylene glycol groups.

“Spirocyclyl” refers to divalent saturated cyclic group from 3 to 10carbon atoms having a cycloalkyl or heterocyclyl ring with a spiro union(the union formed by a single atom which is the only common member ofthe rings) as exemplified by the following structure:

“Sulfo” refers to —SO₃ ⁻ and —SO₃H.

“Sulfoalkyl” refers to -alkyl-SO₃ ⁻ or -alkyl-SO₃H, wherein alkyl isdefined herein.

“Sulfonyl” refers to the divalent group —S(O)₂—.

“Substituted sulfonyl” refers to the group —SO₂-alkyl, —SO₂-substitutedalkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl, —SO₂-cycloalkyl,—SO₂-substituted cycloalkyl, —SO₂-cycloalkenyl, —SO₂-substitutedcycloalkenyl, —SO₂-aryl, —SO₂-substituted aryl, —SO₂-heteroaryl,—SO₂-substituted heteroaryl, —SO₂-heterocyclic, —SO₂-substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic are as defined herein. Substituted sulfonyl includes groupssuch as methyl-SO₂—, phenyl-SO₂—, and 4-methylphenyl-SO₂—.

“Sulfonyloxy” refers to the group —OSO₂-alkyl, —OSO₂-substituted alkyl,—OSO₂-alkenyl, —OSO₂-substituted alkenyl, —OSO₂-cycloalkyl,—OSO₂-substituted cycloalkyl, —OSO₂-cycloalkenyl, —OSO₂-substitutedcylcoalkenyl, —OSO₂-aryl, —OSO₂-substituted aryl, —OSO₂-heteroaryl,—OSO₂-substituted heteroaryl, —OSO₂-heterocyclic, —OSO₂-substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic are as defined herein.

“Thioacyl” refers to the groups H—C(S)—, alkyl-C(S)—, substitutedalkyl-C(S)—, alkenyl-C(S)—, substituted alkenyl-C(S)—, alkynyl-C(S)—,substituted alkynyl-C(S)—, cycloalkyl-C(S)—, substitutedcycloalkyl-C(S)—, cycloalkenyl-C(S)—, substituted cycloalkenyl-C(S)—,aryl-C(S)—, substituted aryl-C(S)—, heteroaryl-C(S)—, substitutedheteroaryl-C(S)—, heterocyclic-C(S)—, and substitutedheterocyclic-C(S)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Thiol” refers to the group —SH.

“Thiocarbonyl” refers to the divalent group —C(S)— which is equivalentto —C(═S)—.

“Thione” refers to the atom (═S).

“Alkylthio” refers to the group —S-alkyl wherein alkyl is as definedherein.

“Substituted alkylthio” refers to the group —S-(substituted alkyl),wherein substituted alkyl is as defined herein.

The term “carrier molecule” as used herein, refers to a biological or anon-biological component that is or becomes covalently bonded to a dyecompound disclosed herein. Such components include, but are not limitedto, an amino acid, a peptide, a protein, a polysaccharide, a nucleoside,a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen,a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer,polyethylene glycol (PEG) groups, a polymeric microparticle, abiological cell, a virus and combinations thereof. Included is oneembodiment in which carrier molecules comprise an organic moiety havingat least 4 plural valent atoms and often more than 10 plural valentatoms (i.e., atoms other than hydrogen and halo), e.g. at least 15 suchatoms, as in the case of moieties having at least 20 such atoms.

The term “conjugated substance” or “Se” refers to a carrier molecule orsolid support.

The term “detectable response” as used herein refers to an occurrence ofor a change in, a signal that is directly or indirectly detectableeither by observation or by instrumentation. Typically, the detectableresponse is an optical response resulting in a change in the wavelengthdistribution patterns or intensity of absorbance or fluorescence or achange in light scatter, fluorescence lifetime, fluorescencepolarization, or a combination of the above parameters.

The term “dye” as used herein refers to a compound that emits light toproduce an observable detectable signal.

As used herein, the term “fluorophore” or “fluorogenic” refers to acompound or a composition that demonstrates a change in fluorescenceupon binding to a biological compound or analyte of interest and/or uponcleavage by an enzyme. The fluorophores of the present disclosure may besubstituted to alter the solubility, spectral properties or physicalproperties of the fluorophore.

As used herein, “a pharmaceutically acceptable salt” or “a biologicallycompatible salt” is a counterion that is not toxic as used, and does nothave a substantially deleterious effect on biomolecules. Examples ofsuch salts include, among others, K⁺, Na⁺, Cs⁺, Li⁺, Ca²⁺, Mg²⁺, Cl⁻,AcO⁻, and alkylammonium or alkoxyammonium salts.

The term “linker” or “L”, as used herein, refers to a single covalentbond or a moiety comprising series of stable covalent bonds, the moietyoften incorporating 1-40 plural valent atoms selected from the groupconsisting of C, N, O, S and P that covalently attach the fluorogenic orfluorescent compounds to another moiety such as a chemically reactivegroup or a biological and non-biological component. The number of pluralvalent atoms in a linker may be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 25, 30 or a larger number up to 40 or more. A linker may belinear or non-linear; some linkers have pendant side chains or pendantfunctional groups, or both. Examples of such pendant moieties arehydrophilicity modifiers, for example solubilizing groups like, e.g.sulfo (—SO₃H or —SO₃). In certain embodiments, L is composed of anycombination of single, double, triple or aromatic carbon-carbon bonds,carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds andcarbon-sulfur bonds. Exemplary linking members include a moiety thatincludes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, and the like. Linkers may,by way of example, consist of a combination of moieties selected fromalkyl; —C(O)NH—; —C(O)O—; —NH—; —S—; —O—; —C(O)—; —S(O)_(n)— where n is0, 1 or 2; —O—; 5- or 6-membered monocyclic rings; and optional pendantfunctional groups, for example sulfo, hydroxy and carboxy. The moietyformed by a linker bonded to a reactive group (R_(x)) may be designated-L-R_(x). The reactive group may be reacted with a substance reactivetherewith, whereby the linker becomes bonded to a conjugated substance(S_(c)) and may be designated -L-S_(c), or in some cases, the linker maycontains a residue of a reactive group (e.g. the carbonyl group of anester) and may be designated “-L_(R)”. A “cleavable linker” is a linkerthat has one or more cleavable groups that may be broken by the resultof a reaction or condition. The term “cleavable group” refers to amoiety that allows for release of a portion, e.g., a fluorogenic orfluorescent moiety, of a conjugate from the remainder of the conjugateby cleaving a bond linking the released moiety to the remainder of theconjugate. Such cleavage is either chemical in nature, or enzymaticallymediated. Exemplary enzymatically cleavable groups include natural aminoacids or peptide sequences that end with a natural amino acid.

In addition to enzymatically cleavable groups, it is within the scope ofthe present invention to include one or more sites that are cleaved bythe action of an agent other than an enzyme. Exemplary non-enzymaticcleavage agents include, but are not limited to, acids, bases, light(e.g., nitrobenzyl derivatives, phenacyl groups, benzoin esters), andheat. Many cleavable groups are known in the art. See, for example, Junget al., Biochem. Biophys. Acta. 761:152-162 (1983); Joshi et al., J.Biol. Chem., 265:14518-14525 (1990); Zarling et al., J. Immunol.,124:913-920 (1980); Bouizar et al., Eur. J. Biochem., 155:141-147(1986); Park et al., J. Biol. Chem., 261:205-210 (1986); Browning etal., J. Immunol., 143:1859-1867 (1989). Moreover a broad range ofcleavable, bifunctional (both homo- and hetero-bifunctional) spacer armsare commercially available.

An exemplary cleavable group, such as an ester, is cleavable group thatmay be cleaved by a reagent, e.g., sodium hydroxide, resulting in acarboxylate-containing fragment and a hydroxyl-containing product.

The linker may be used to attach the dye compound to another componentof a conjugate, such as a targeting moiety (e.g., antibody, ligand,non-covalent protein-binding group, etc.), an analyte, a biomolecule, adrug and the like.

The term “reactive group” (or “R_(x)”), as used herein, refers to agroup that is capable of reacting with another chemical group to form acovalent bond, i.e., is covalently reactive under suitable reactionconditions, and generally represents a point of attachment for anothersubstance. The reactive group is a moiety, such as carboxylic acid orsuccinimidyl ester, on the compounds of the present disclosure that iscapable of chemically reacting with a functional group on a differentcompound to form a covalent linkage. Reactive groups generally includenucleophiles, electrophiles and photoactivatable groups.

Exemplary reactive groups include, but not limited to, olefins,acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes,ketones, carboxylic acids, esters, amides, cyanates, isocyanates,thiocyanates, isothiocyanates, amines, hydrazines, hydrazones,hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides,disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids,acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles,amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamicacids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines,ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates,imines, azides, azo compounds, azoxy compounds, and nitroso compounds.Reactive functional groups also include those used to preparebioconjugates, e.g., N-hydroxysuccinimide esters, maleimides,succinimidyl esters (SE), sulfodichlorophenyl (SDP) esters,sulfotetrafluorophenyl (STP) esters, tetrafluorophenyl (TFP) esters,pentafluorophenyl (PFP) esters, nitrilotriacetic acids (NTA),aminodextrans, cyclooctyne-amines and the like. Methods to prepare eachof these functional groups are well known in the art and theirapplication to or modification for a particular purpose is within theability of one of skill in the art (see, for example, Sandler and Karo,eds., Organic Functional Group Preparations, Academic Press, San Diego,1989).

The term “solid support,” as used herein, refers to a matrix or mediumthat is substantially insoluble in liquid phases and capable of bindinga molecule or particle of interest.

Solid supports suitable for use herein include semi-solid supports andare not limited to a specific type of support. Useful solid supportsinclude solid and semi-solid matrixes, such as aerogels and hydrogels,resins, beads, biochips (including thin film coated biochips),microfluidic chip, a silicon chip, multi-well plates (also referred toas microtitre plates or microplates), membranes, conducting andnonconducting metals, glass (including microscope slides) and magneticsupports. More specific examples of useful solid supports include silicagels, polymeric membranes, particles, derivatized plastic films, glassbeads, cotton, plastic beads, alumina gels, polysaccharides such asSEPHAROSE (GE Healthcare), poly(acrylate), polystyrene,poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch,FICOLL (GE Healthcare), heparin, glycogen, amylopectin, mannan, inulin,nitrocellulose, diazocellulose, polyvinylchloride, polypropylene,polyethylene (including poly(ethylene glycol)), nylon, latex bead,magnetic bead, paramagnetic bead, superparamagnetic bead, starch and thelike.

Compounds and Compositions:

In general, for ease of understanding the present disclosure, compoundsand corresponding substituents will first be described in detail,followed by various methods in which the compounds of the presentdisclosure are useful, which is followed by exemplary methods of use ofcertain compounds that are particularly advantageous for use with themethods provided herein.

The compounds disclosed herein are useful for short- and long-termtracking of cell proliferation, differentiation and/or function, as wellas for staining samples, labeling proteins, detecting ligands and otheranalytes.

Cell movement and location studies require detectable compounds that arenon-toxic to living cells and are available in a range of fluorescentcolors to match instrument lasers and filters and to accommodateco-staining with antibodies or other cell analysis compounds. Thecompounds described herein are useful for monitoring cell movement,location, proliferation, migration, chemotaxis and invasion. Thecompounds described herein can pass freely through the cell membrane;however, once inside the cell, they are transformed into cell-impermeantreaction products and are well retained in living cells over severalgenerations. The compounds described herein are transferred to daughtercells, but not to adjacent cells in a population. The compoundsdisclosed herein have the following advantages: they have a highsignal:noise (S:N) ratio by imaging and flow cytometry; they arenon-toxic to cells thereby allowing for tracking of multiple celldivisions; they are highly cell permeable; and they are brightlyfluorescent at physiological pH.

The compounds described herein can permanently label cells withoutaffecting the cell's morphology or physiology in order to tracegenerations or divisions both in vivo and in vitro. The bright,single-peak staining of the compounds described herein enablesvisualization of multiple generations with long-term signal stability.The compounds described herein are well retained within cells forseveral days post-stain. The compounds are non-cytotoxic, meaning thatthey have no known effect on the proliferation ability or biology of thecells. The compounds described herein are reactive fluorescent moleculeswhich enter the cells freely via diffusion. Upon entering the cell, thecompounds react with intracellular proteins thereby allowing theconjugated compounds to be retained within the cells. Daughter cellsreceive approximately half of the label from the parent. Analysis of thelevel of fluorescence in the cell population by flow cytometry, forexample, permits determination of the number of generations throughwhich the cell has progressed since the label was applied.

One illustrative embodiment provides a compound having structuralformula (I):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵, and R¹⁶ are eachindependently H, halogen, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c); or

R⁷ taken together with R¹⁴ are part of an optionally substituted 5- or6-membered ring;

R⁸ taken together with R¹⁵ are part of an optionally substituted 5- or6-membered ring;

R⁹ taken together with R¹¹ are part of an optionally substituted 5- or6-membered ring;

R¹⁰ taken together with R¹⁶ are part of an optionally substituted 5- or6-membered ring;

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance.

In certain embodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). Incertain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are each independently H,alkyl, substituted alkyl, or sulfoalkyl. In certain embodiments, R⁷, R⁸,R⁹, and R¹⁰ are each independently a C₁-C₆ alkyl, which can be the sameor different. In certain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are eachmethyl. In certain embodiments, R¹² and R¹³ are each alkyl, preferably aC₁-C₆ alkyl. In certain embodiments, R¹² and R¹³ are each methyl. Incertain embodiments, R¹¹ and R¹⁴ are each independently alkyl,substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certainembodiments, R¹¹ and R¹⁴ are each independently a C₁-C₆ alkyl, a C₁-C₆substituted alkyl, or sulfoalkyl, which can be the same or different. Incertain embodiments, R_(x) is succinimidyl ester (SE),sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP) ester,tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another illustrative embodiment provides a compound having structuralformula (II):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹ and R²²are each independently H, halogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R³, R⁴, R⁵ and R⁶ are each independently H or halo. Incertain embodiments, R³, R⁴, R⁵ and R⁶ are independently H, Cl, or F. Incertain embodiments, R⁷ and R⁹ are each independently alkyl, substitutedalkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certain embodiments,R⁷ and R⁹ are each independently methyl, -L-R_(x), -L-S_(c) or(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 3. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another illustrative embodiment provides a compound having structuralformula (III):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³,and R²⁴ are each independently H, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R⁷, R⁹, R¹⁷, R¹⁹, R²⁰, R²², R²³ and R²⁴ are eachindependently alkyl, substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or-L-S_(c). In certain embodiments, R⁷, R⁹, R¹⁷, R²², R²³ and R²⁴ are eachmethyl. In certain embodiments, R¹⁹ and R²⁰ are each independently(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 1. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another illustrative embodiment provides a compound having structuralformula (IV):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²¹, R²², R²³, andR²⁴ are each independently H, halogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance.

In certain embodiments, R⁹, R¹², R¹³, R²² and R²³ are each alkyl,preferably a C₁-C₆ alkyl.

In certain embodiments, R⁹, R¹², R¹³, R²² and R²³ are each methyl. Incertain embodiments, R³, R⁴, R⁵ and R⁶ are each independently H or halo.In certain embodiments, R³ and R⁶ are independently H, Cl, or F. Incertain embodiments, R² is carboxyl. In certain embodiments, R⁷, R⁹ andR²⁰ are each independently alkyl, substituted alkyl, sulfoalkyl, sulfo,(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6, -L-R_(x), or-L-S_(c). In certain embodiments, R⁷ is -L-R_(x) and R²⁰ is (CH₂)_(n)SO₃⁻. In certain embodiments, n is 1. In certain embodiments, R_(x) issuccinimidyl ester (SE), sulfodichlorophenyl (SDP) ester,sulfotetrafluorophenyl (STP) ester, tetrafluorophenyl (TFP) ester,pentafluorophenyl (PFP) ester, nitrilotriacetic acid (NTA),aminodextran, and cyclooctyne-amine.

In certain embodiments, R_(x) is selected from an acrylamide, anactivated ester of a carboxylic acid, a carboxylic ester, an acyl azide,an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline,an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a photoactivatable group, a reactive platinum complex,a silyl halide, a sulfonyl halide, and a thiol. In certain embodimentsthe reactive group is selected from the group consisting of carboxylicacid, succinimidyl ester of a carboxylic acid, hydrazide, amine and amaleimide. The reactive group may be attached to any appropriate site onthe reporter molecule or the aniline moiety. In certain embodiments, atleast one member selected from R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹²,R¹³, R¹⁹ and R²⁰ is a reactive group. In certain embodiments, at leastone of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is a reactive group. Incertain embodiments, at least one of R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is areactive group.

In certain embodiments, S_(c) is selected from a carrier molecule and asolid support. In certain embodiments, S_(c) is selected from an aminoacid, a polymer of amino acids, a peptide, a protein, a neurotoxin, aphallotoxin, a cytokine, a toxin, a protease substrate, a protein kinasesubstrate, an enzyme, an antibody, an antibody fragment, a lectin, aglycoprotein, a histone, an albumin, a lipoprotein, avidin,streptavidin, protein A, protein G, a phycobiliprotein, a fluorescentprotein, a hormone, a growth factor, a nucleic acid base, a nucleoside,a nucleotide, a nucleic acid polymer, a nucleotide analog, a nucleosideanalog, a nucleoside triphosphate, a deoxynucleoside triphosphate, adideoxynucleoside triphosphate, a hapten, a carbohydrate, apolysaccharide, a lipid, an ion-complexing moiety (such as a crownether), a PEG group, and an organic or inorganic polymer. In certainembodiments, at least one member selected from R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁹ and R²⁰ is a conjugated substance. In certainembodiments, at least one of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ isa conjugated substance. In certain embodiments, at least one of R⁷, R⁸,R⁹, R¹⁰, R¹² and R¹³ is a conjugated substance. In certain embodiments,at least one of R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴,R¹⁵ and R¹⁶ is a PEG group.

In certain embodiments, one or more PEG groups are added to thecarbopyronine compounds. Ethylene glycol, diethylene glycol andpolyethylene glycol are herein collectively referred to as PEG. The PEGgroups are either directly or indirectly linked to the compounds.Indirect attachment indicates the use of a linker. Direct attachmentindicates lack of a linker.

In certain embodiments, compounds are provided selected from the groupconsisting of:

forms thereof.

Reactive Groups:

In certain embodiments, the compounds provided herein are chemicallyreactive, and are substituted by at least one reactive group (R_(x)).The reactive group functions as the site of attachment for anothermoiety, such as a carrier molecule or a solid support, wherein thereactive group chemically reacts with an appropriate reactive orfunctional group on the carrier molecule or solid support. Thus, incertain embodiments, the compounds provided herein comprise an anilinemoiety, linker, fluorophore, a reactive group moiety and optionally acarrier molecule and/or a solid support.

In certain embodiments, the compounds provided herein further comprise areactive group which is a member selected from an acrylamide, anactivated ester of a carboxylic acid, a carboxylic ester, an acyl azide,an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline,an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a photoactivatable group, a reactive platinum complex,a silyl halide, a sulfonyl halide, and a thiol. In certain embodimentsthe reactive group is selected from the group consisting of carboxylicacid, succinimidyl ester of a carboxylic acid, hydrazide, amine and amaleimide. The reactive group may be attached to any appropriate site onthe reporter molecule or the aniline moiety. In certain embodiments, atleast one member selected from R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹²,R¹³, R¹⁹ and R²⁰ is a reactive group. In certain embodiments, at leastone of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is a reactive group. Incertain embodiments, at least one of R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is areactive group. Alternatively, if the compounds disclosed hereincomprise a carrier molecule or solid support a reactive group may becovalently attached independently to those substituents, allowing forfurther conjugation to a fluorophore, carrier molecule or solid support.

These reactive groups are synthesized during the formation of thecompounds provided herein and carrier molecule- and/or solidsupport-containing compounds to provide chemically reactive compounds.In this way, compounds incorporating a reactive group may be covalentlyattached to a wide variety of carrier molecules or solid supports thatcontain, or are modified to contain, functional groups with suitablereactivity, resulting in chemical attachment of the components. Incertain embodiments, the reactive group of the compounds disclosedherein and the functional group of the carrier molecule or solid supportcomprise electrophiles and nucleophiles that can generate a covalentlinkage between them. In certain embodiments, the reactive groupcomprises a photoactivatable group, which becomes chemically reactiveonly after illumination with light of an appropriate wavelength.Typically, the conjugation reaction between the reactive group and thecarrier molecule or solid support results in one or more atoms of thereactive group being incorporated into a new linkage attaching the dyecompounds disclosed herein to the carrier molecule or solid support.Selected examples of functional groups and linkages are shown in Table1, where the reaction of an electrophilic group and a nucleophilic groupyields a covalent linkage.

TABLE 1 Examples of some routes to useful covalent linkagesElectrophilic Group Nucleophilic Group Resulting Covalent Linkageactivated esters* amines/anilines carboxamides acrylamides thiolsthioethers acyl azides** amines/anilines carboxamides acyl halidesamines/anilines carboxamides acyl halides alcohols/phenols esters acylnitriles alcohols/phenols esters acyl nitriles amines/anilinescarboxamides aldehydes amines/anilines imines aldehydes or ketoneshydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkylhalides amines/anilines alkyl amines alkyl halides carboxylic acidsesters alkyl halides thiols thioethers alkyl halides alcohols/phenolsethers alkyl sulfonates thiols thioethers alkyl sulfonates carboxylicacids esters alkyl sulfonates alcohols/phenols ethers anhydridesalcohols/phenols esters anhydrides amines/anilines carboxamides arylhalides thiols thiophenols aryl halides amines aryl amines aziridinesthiols thioethers boronates glycols boronate esters carbodiimidescarboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic acidsesters epoxides thiols thioethers haloacetamides thiols thioethershaloplatinate amino platinum complex haloplatinate heterocycle platinumcomplex haloplatinate thiol platinum complex halotriazinesamines/anilines aminotriazines halotriazines alcohols/phenols triazinylethers halotriazines thiols triazinyl thioethers imido estersamines/anilines amidines isocyanates amines/anilines ureas isocyanatesalcohols/phenols urethanes isothiocyanates amines/anilines thioureasmaleimides thiols thioethers phosphoramidites alcohols phosphite esterssilyl halides alcohols silyl ethers sulfonate esters amines/anilinesalkyl amines sulfonate esters thiols thioethers sulfonate esterscarboxylic acids esters sulfonate esters alcohols ethers sulfonylhalides amines/anilines sulfonamides sulfonyl halides phenols/alcoholssulfonate esters *Activated esters, as understood in the art, generallyhave the formula —COΩ, where Ω is a suitable leaving group (e.g.,succinimidyloxy (—OC₄H₄O₂), sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H),-1-oxybenzotriazolyl (—OC₆H₄N₃); or an aryloxy group or aryloxysubstituted one or more times by electron withdrawing substituents suchas nitro, fluoro, chloro, cyano, or trifluoromethyl, or combinationsthereof, used to form activated aryl esters; or a carboxylic acidactivated by a carbodiimide to form an anhydride or mixed anhydride—OCOR^(x) or —OCNR^(x)NHR^(y), where R^(x) and R^(y), which may be thesame or different, are C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, or C₁-C₆alkoxy; or cyclohexyl, 3-dimethylaminopropyl, or N-morpholinoethyl).**Acyl azides can also rearrange to isocyanates.

The choice of the reactive group used to attach the dye compoundsdisclosed herein to the substance to be conjugated typically depends onthe reactive or functional group on the substance to be conjugated andthe type or length of covalent linkage desired. The types of functionalgroups typically present on the organic or inorganic substances(biomolecule or non-biomolecule) include, but are not limited to,amines, amides, thiols, alcohols, phenols, aldehydes, ketones,phosphates, imidazoles, hydrazines, hydroxylamines, disubstitutedamines, halides, epoxides, silyl halides, carboxylate esters, sulfonateesters, purines, pyrimidines, carboxylic acids, olefinic bonds, or acombination of these groups. A single type of reactive site may beavailable on the substance (typical for polysaccharides or silica), or avariety of sites may occur (e.g., amines, thiols, alcohols, phenols), asis typical for proteins.

Typically, the reactive group will react with an amine, a thiol, analcohol, an aldehyde, a ketone, or with silica. Preferably, reactivegroups react with an amine or a thiol functional group, or with silica.In certain embodiments, the reactive group is an acrylamide, anactivated ester of a carboxylic acid, an acyl azide, an acyl nitrile, analdehyde, an alkyl halide, a silyl halide, an anhydride, an aniline, anaryl halide, an azide, an aziridine, a boronate, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine (including hydrazides), animido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a sulfonyl halide, or athiol group. As used herein, “reactive platinum complex” refers tochemically reactive platinum complexes such as described in U.S. Pat.No. 5,714,327, herein incorporated by reference in its entirety.

In certain embodiments, the compounds disclosed herein comprise at leastone reactive group that selectively reacts with an amine group. Thisamine-reactive group is selected from the group consisting ofsuccinimidyl ester (SE), sulfonyl halide, tetrafluorophenyl (TFP) ester,sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP) ester,pentafluorophenyl (PFP) ester and iosothiocyanates. Thus, in certainembodiments, the compounds provided herein form a covalent bond with anamine containing molecule in a sample. In certain embodiments, thecompounds provided herein comprise at least one reactive group thatselectively reacts with a thiol group. This thiol-reactive group isselected from the group consisting of maleimide, haloalkyl andhaloacetamide (including any reactive groups disclosed in U.S. Pat. Nos.5,362,628; 5,352,803 and 5,573,904, all of which are herein incorporatedby reference in their entirety).

Where the reactive group is an activated ester of a carboxylic acid,such as a succinimidyl ester of a carboxylic acid, a sulfonyl halide,tetrafluorophenyl (TFP) ester, sulfodichlorophenyl (SDP) ester,sulfotetrafluorophenyl (STP) ester, pentafluorophenyl (PFP) ester or anisothiocyanate, the resulting compound is particularly useful forpreparing conjugates of carrier molecules such as proteins, nucleotides,oligonucleotides, or haptens. Where the reactive group is a maleimide,haloalkyl or haloacetamide (including any reactive groups disclosed inU.S. Pat. Nos. 5,362,628; 5,352,803 and 5,573,904, all of which areherein incorporated by reference in their entirety) the resultingcompound is particularly useful for conjugation to thiol-containingsubstances. Where the reactive group is a hydrazide, the resultingcompound is particularly useful for conjugation to periodate-oxidizedcarbohydrates and glycoproteins, and in addition is an aldehyde-fixablepolar tracer for cell microinjection. Where the reactive group is asilyl halide, the resulting compound is particularly useful forconjugation to silica surfaces, particularly where the silica surface isincorporated into a fiber optic probe subsequently used for remote iondetection or quantitation.

In a certain embodiments, the reactive group is a photoactivatable groupsuch that the group is only converted to a reactive species afterillumination with an appropriate wavelength. An appropriate wavelengthis generally a UV wavelength that is less than 400 nm. This methodprovides for specific attachment to only the target molecules, either insolution or immobilized on a solid or semi-solid matrix.Photoactivatable reactive groups include, without limitation,benzophenones, aryl azides and diazirines.

Preferably, the reactive group is a photoactivatable group, succinimidylester of a carboxylic acid, a haloacetamide, haloalkyl, a hydrazine, anisothiocyanate, a maleimide group, an aliphatic amine, a silyl halide, acadaverine or a psoralen. More preferably, the reactive group is asuccinimidyl ester of a carboxylic acid, a maleimide, an iodoacetamide,or a silyl halide. In certain embodiments, the reactive group is asuccinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester, an iosothiocyanates or a maleimide. In certainembodiments, the reactive group is selected from sulfodichlorophenyl(SDP) ester, sulfotetrafluorophenyl (STP) ester, tetrafluorophenyl (TFP)ester, a pentafluorophenyl (PFP) ester, and a nitrilotriacetic acid(NTA).

Carrier Molecules:

In certain embodiments, the compounds provided herein are covalentlybound to a carrier molecule. If the compound has a reactive group, thenthe carrier molecule may alternatively be linked to the compound throughthe reactive group. The reactive group may contain both a reactivefunctional moiety and a linker, or only the reactive functional moiety.

A variety of carrier molecules are useful herein. Exemplary carriermolecules include antigens, steroids, vitamins, drugs, haptens,metabolites, toxins, environmental pollutants, amino acids, peptides,proteins, nucleic acids, nucleic acid polymers, carbohydrates, lipids,polymers and bacterial particles. In certain embodiments, at least onemember selected from R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁹and R²⁰ is a carrier molecule. In certain embodiments, at least one ofR², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is a carrier molecule. Incertain embodiments, at least one of R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is acarrier molecule.

In certain embodiments, the carrier molecule comprises an amino acid, apeptide, a protein, a polysaccharide, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, ahormone, a lipid, a lipid assembly, a synthetic polymer, a polymericmicroparticle, a biological cell, a virus and combinations thereof. Incertain embodiments, the carrier molecule is selected from a hapten, anucleotide, an oligonucleotide, a nucleic acid polymer, a protein, apeptide or a polysaccharide. In certain embodiments the carrier moleculeis amino acid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a tyramine, a syntheticpolymer, a polymeric microparticle, a biological cell, cellularcomponents, an ion chelating moiety, an enzymatic substrate or a virus.In certain embodiments, the carrier molecule is an antibody or fragmentthereof, an antigen, an avidin or streptavidin, a biotin, a dextran, anIgG binding protein, a fluorescent protein, agarose, and anon-biological microparticle. In certain embodiments, carrier moleculesmay comprise a label or a fluorescent dye or quencher.

In certain embodiments, the carrier molecule is an amino acid (includingthose that are protected or are substituted by phosphates,carbohydrates, or C₁ to C₂₂ carboxylic acids), or a polymer of aminoacids such as a peptide or protein. In certain embodiments, the carriermolecule contains at least five amino acids, more preferably 5 to 36amino acids. Exemplary peptides include, but are not limited to,neuropeptides, cytokines, toxins, protease substrates, and proteinkinase substrates. Other exemplary peptides may function as organellelocalization peptides, that is, peptides that serve to target theconjugated compound for localization within a particular cellularsubstructure by cellular transport mechanisms. Preferred protein carriermolecules include enzymes, antibodies, lectins, glycoproteins, histones,albumins, lipoproteins, avidin, streptavidin, protein A, protein G,phycobiliproteins and other fluorescent proteins, hormones, toxins andgrowth factors. Typically, the protein carrier molecule is an antibody,an antibody fragment, avidin, streptavidin, a toxin, a lectin, a growthfactor, bacterial particle or a binding partner for a cell receptor.

In certain embodiments, the carrier molecule comprises a nucleic acidbase, nucleoside, nucleotide or a nucleic acid polymer, optionallycontaining an additional linker or spacer for attachment of afluorophore or other ligand, such as an alkynyl linkage (U.S. Pat. No.5,047,519), an aminoallyl linkage (U.S. Pat. No. 4,711,955) or otherlinkage. In certain embodiments, the nucleotide carrier molecule is anucleoside or a deoxynucleoside or a dideoxynucleoside.

Exemplary nucleic acid polymer carrier molecules are single- ormulti-stranded, natural or synthetic DNA or RNA oligonucleotides, orDNA/RNA hybrids, or incorporating an unusual linker such as morpholinederivatized phosphates (AntiVirals, Inc., Corvallis Oreg.), or peptidenucleic acids such as N-(2-aminoethyl)glycine units, where the nucleicacid contains fewer than 50 nucleotides, more typically fewer than 25nucleotides.

In certain embodiments, the carrier molecule comprises a carbohydrate orpolyol that is typically a polysaccharide, such as dextran, FICOLL(GEHealthcare), heparin, glycogen, amylopectin, mannan, inulin, starch,agarose and cellulose, or is a polymer such as a poly(ethylene glycol).In certain embodiments, the polysaccharide carrier molecule includesdextran, agarose or FICOLL. In certain embodiments, the carrier moleculeincludes a PEG group.

In certain embodiments, the carrier molecule comprises a lipid(typically having 6-25 carbons), including glycolipids, phospholipids,and sphingolipids. In certain embodiments, the carrier moleculecomprises a lipid vesicle, such as a liposome, or is a lipoprotein. Somelipophilic substituents are useful for facilitating transport of theconjugated dye into cells or cellular organelles.

In certain embodiments, the carrier molecule is a cell, cellular system,cellular fragment, or subcellular particles, including virus particles,bacterial particles, virus components, biological cells (such as animalcells, plant cells, bacteria, or yeast), or cellular components.Examples of cellular components that are useful as carrier moleculesinclude lysosomes, endosomes, cytoplasm, nuclei, histones, mitochondria,Golgi apparatus, endoplasmic reticulum and vacuoles.

In certain embodiments, the carrier molecule non-covalently associateswith organic or inorganic materials. Exemplary embodiments of thecarrier molecule that possess a lipophilic substituent may be used totarget lipid assemblies such as biological membranes or liposomes bynon-covalent incorporation of the compound within the membrane, e.g.,for use as probes for membrane structure or for incorporation inliposomes, lipoproteins, films, plastics, lipophilic microspheres orsimilar materials.

In certain embodiments, the carrier molecule comprises a specificbinding pair member wherein the compounds provided herein are conjugatedto a specific binding pair member and used to the formation of the boundpair. Alternatively, the presence of the labeled specific binding pairmember indicates the location of the complementary member of thatspecific binding pair; each specific binding pair member having an areaon the surface or in a cavity which specifically binds to, and iscomplementary with, a particular spatial and polar organization of theother. In this instance, the dye compounds disclosed herein function asa reporter molecule for the specific binding pair. Exemplary bindingpairs are set forth in Table 2.

TABLE 2 Representative Specific Binding Pairs Antigen Antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor folate folate binding protein toxin toxin receptorcarbohydrate lectin or carbohydrate receptor peptide peptide receptorprotein protein receptor enzyme substrate enzyme DNA (RNA) cDNA(cRNA)^(†) hormone hormone receptor ion chelator *IgG is animmunoglobulin ^(†)cDNA and cRNA are the complementary strands used forhybridization

Solid Supports:

In certain embodiments, the compounds disclosed herein are covalentlybonded to a solid support. The solid support may be attached to thecompounds either through the aniline moiety, fluorophore, or through areactive group, if present, or through a carrier molecule, if present.Even if a reactive group and/or a carrier molecule are present, thesolid support may be attached through the aniline moiety or fluorophore.In certain embodiments, at least one member selected from R², R³, R⁴,R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁹ and R²⁰ is a solid support. Incertain embodiments, at least one of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹²and R¹³ is a solid support. In certain embodiments, at least one of R⁷,R⁸, R⁹, R¹⁰, R¹² and R¹³ is a solid support.

Solid supports suitable for use herein are typically substantiallyinsoluble in liquid phases. Solid supports for use herein are notlimited to a specific type of support. Rather, a large number ofsupports are available and are known to one of ordinary skill in theart. Thus, useful solid supports include solid and semi-solid matrixes,such as aerogels and hydrogels, resins, beads, biochips (including thinfilm coated biochips), microfluidic chip, a silicon chip, multi-wellplates (also referred to as microtitre plates or microplates),membranes, conducting and nonconducting metals, glass (includingmicroscope slides) and magnetic supports. More specific examples ofuseful solid supports include silica gels, polymeric membranes,particles, derivatized plastic films, glass beads, cotton, plasticbeads, alumina gels, polysaccharides such as SEPHAROSE (GE Healthcare),poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar,cellulose, dextran, starch, FICOLL (GE Healthcare), heparin, glycogen,amylopectin, mannan, inulin, nitrocellulose, diazocellulose,polyvinylchloride, polypropylene, polyethylene (including poly(ethyleneglycol)), nylon, latex bead, magnetic bead, paramagnetic bead,superparamagnetic bead, starch and the like.

In certain embodiments, the solid support may include a solid supportreactive functional group, including, but not limited to, hydroxyl,carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea,carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide,sulfoxide, etc., for attaching the dye compounds disclosed herein.Useful reactive groups are disclosed above and are equally applicable tothe solid support reactive functional groups herein.

A suitable solid phase support may be selected on the basis of desiredend use and suitability for various synthetic protocols. For example,where amide bond formation is desirable to attach the compoundsdisclosed herein to the solid support, resins generally useful inpeptide synthesis may be employed, such as polystyrene (e.g., PAM-resinobtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE resin(obtained from Aminotech, Canada), polyamide resin (obtained fromPeninsula Laboratories), polystyrene resin grafted with polyethyleneglycol (TENTAGEL, Rapp Polymere, Tubingen, Germany),polydimethyl-acrylamide resin (available from Milligen/Biosearch,California), or PEGA beads (obtained from Polymer Laboratories).

Preparation of Conjugates:

Another illustrative embodiment provides a process for preparing aconjugated compound of structural formula (I), (II), (III) or (IV) theprocess comprising:

reacting a compound of structural formula (I), (II), (III) or (IV) witha substance to be conjugated thereto, thereby resulting in a conjugatedsubstance S_(C).

The compounds disclosed herein that contain a reactive group R_(X) areuseful to fluorescently label a wide variety of organic substances thatcontain functional groups with suitable reactivity, resulting inchemical attachment, i.e., conjugation, of the substance (therebyaffording a conjugated substance, S_(C)) and formation of compounds thatare themselves conjugates. Most preferably, but not exclusively, theconjugated substance disclosed herein is an intracellular amino acid,peptide, protein, nucleotide, oligonucleotide, nucleic acid, lipid,phospholipid, lipoprotein, or lipopolysaccharide. The reactive group andfunctional group are typically an electrophile and a nucleophile,respectively, that can generate a covalent linkage. Alternatively, thereactive group is a photoactivatable group that becomes chemicallyreactive only after illumination with light of an appropriatewavelength. Selected examples of functional groups and linkages, wherethe reaction of an electrophilic group and a nucleophilic group yields acovalent linkage, as well as a general discussion of dye-conjugatechemistry, are provided in U.S. Pat. No. 5,830,912 the disclosure ofwhich is incorporated herein by reference in its entirety.

In certain embodiments, conjugates of the compounds disclosed herein areprovided. One or more of the compounds provided herein are conjugated toa biologically compatible polymer, including amino acid polymers(typically proteins, including IgG antibodies), carbohydrate polymers(typically dextrans), and polymeric microspheres (typically polystyrene)and are readily prepared for use as tracers according to methods knownin the art.

Conjugates of components (carrier molecules or solid supports), e.g.,drugs, peptides, toxins, nucleotides, phospholipids, proteins and otherorganic molecules are prepared by organic synthesis methods using thecompounds disclosed herein, are generally prepared by means wellrecognized in the art (Haugland, MOLECULAR PROBES HANDBOOK, supra,(2002)). Preferably, conjugation to form a covalent bond consists ofmixing the compounds disclosed herein in a suitable solvent in whichboth the compound and the substance to be conjugated are soluble. Thereaction preferably proceeds spontaneously without added reagents atroom temperature or below. For those reactive compounds that arephotoactivated, conjugation is facilitated by illumination of thereaction mixture to activate the reactive compound. Chemicalmodification of water-insoluble substances, so that a desiredcompound-conjugate may be prepared, is preferably performed in anaprotic solvent such as dimethylformamide, dimethylsulfoxide, acetone,ethyl acetate, toluene, or chloroform. Similar modification ofwater-soluble materials is readily accomplished through the use of theinstant reactive compounds to make them more readily soluble in organicsolvents.

Preparation of peptide or protein conjugates typically comprises firstdissolving the protein to be conjugated in aqueous buffer at about 1-10mg/mL at room temperature or below. Bicarbonate buffers (pH about 8.3)are especially suitable for reaction with succinimidyl esters, phosphatebuffers (pH about 7.2-8) for reaction with thiol-reactive functionalgroups and carbonate or borate buffers (pH about 9) for reaction withisothiocyanates and dichlorotriazines. The appropriate reactive compoundis then dissolved in a nonhydroxylic solvent (usually DMSO or DMF) in anamount sufficient to give a suitable degree of conjugation when added toa solution of the protein to be conjugated. The appropriate amount ofcompound for any protein or other component is convenientlypredetermined by experimentation in which variable amounts of the dyecompound are added to the protein, the conjugate is chromatographicallypurified to separate unconjugated dye compound and the dyecompound-protein conjugate is tested in its desired application.

Following addition of the compound to the component solution, themixture is incubated for a suitable period (typically about 1 hour atroom temperature to several hours on ice), the excess compound isremoved by gel filtration, dialysis, HPLC, adsorption on an ion exchangeor hydrophobic polymer or other suitable means. The dyecompound-conjugate may be used in solution or lyophilized. In this way,suitable conjugates may be prepared from antibodies, antibody fragments,avidins, lectins, enzymes, proteins A and G, cellular proteins,albumins, histones, growth factors, hormones, and other proteins.

Conjugates of polymers, including biopolymers and other higher molecularweight polymers are typically prepared by means well recognized in theart (for example, Brinkley et al., Bioconjugate Chem., 3:2 (1992)). Inthese embodiments, a single type of reactive site may be available, asis typical for polysaccharides) or multiple types of reactive sites(e.g. amines, thiols, alcohols, phenols) may be available, as is typicalfor proteins. Selectivity of labeling is best obtained by selection ofan appropriate reactive dye compound. For example, modification ofthiols with a thiol-selective reagent such as a haloacetamide ormaleimide, or modification of amines with an amine-reactive reagent suchas an activated ester, acyl azide, isothiocyanate or3,5-dichloro-2,4,6-triazine. Partial selectivity may also be obtained bycareful control of the reaction conditions.

When modifying polymers with the compounds disclosed herein, an excessof compound is typically used, relative to the expected degree of dyecompound substitution. Any residual, unreacted compound or a compoundhydrolysis product is typically removed by dialysis, chromatography orprecipitation. Presence of residual, unconjugated compound may bedetected by thin layer chromatography using a solvent that elutes thecompound away from its conjugate. In all cases it is usually preferredthat the reagents be kept as concentrated as practical so as to obtainadequate rates of conjugation.

In certain embodiments, the conjugates of the compounds disclosed hereinare associated with an additional substance, that binds either to thefluorophore or the conjugated substance (carrier molecule or solidsupport) through non-covalent interaction. In another exemplaryembodiment, the additional substance is an antibody, an enzyme, ahapten, a lectin, a receptor, an oligonucleotide, a nucleic acid, aliposome, or a polymer. The additional substance is optionally used toprobe for the location of the conjugate, for example, as a means ofenhancing the signal of the conjugate.

In certain embodiments, compositions are provided, the compositionscomprising:

a) one or more of the compounds provided herein; and

b) a carrier.

In certain embodiments, compositions are provided, the compositionscomprising:

a) one or more of the compounds provided herein; and

b) an analyte.

In certain embodiments, compositions are provided, the compositionscomprising:

a) one or more of the compounds of structural formula (I), (II), (III)or (IV); and

b) a carrier,

wherein the one or more of the compounds are present in an amounteffective to track cell proliferation, differentiation, and/or function.

In certain embodiments, compositions are provided, the compositionscomprising:

(a) one or more of the compounds of structural formula (I), (II), (III)or (IV); and

(b) an analyte,

wherein the one or more of the compounds are present in an amounteffective to track cell proliferation, differentiation, and/or function.

In certain embodiments, the analyte is a cell and the compound islocated inside the cell. In certain embodiments, the compound isconjugated to a carrier molecule.

Methods:

Another embodiment provides a method for tracking cell proliferation,differentiation, and/or function, the method being compatible for usewith, for example, flow cytometry and fluorescence microscopy, andcomprising:

a) incubating a mixture of cells and a compound of structural formula(I):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵, and R¹⁶ are eachindependently H, halogen, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c); or

R⁷ taken together with R¹⁴ are part of an optionally substituted 5- or6-membered ring;

R⁸ taken together with R¹⁵ are part of an optionally substituted 5- or6-membered ring;

R⁹ taken together with R¹¹ are part of an optionally substituted 5- or6-membered ring;

R¹⁰ taken together with R¹⁶ are part of an optionally substituted 5- or6-membered ring;

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) providing a stimulus to the mixture to elicit a fluorescent signal;and

c) analyzing the stimulated mixture.

In certain embodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). Incertain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are each independently H,alkyl, substituted alkyl, or sulfoalkyl. In certain embodiments, R⁷, R⁸,R⁹, and R¹⁰ are each independently a C₁-C₆ alkyl, which can be the sameor different. In certain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are eachmethyl. In certain embodiments, R¹² and R¹³ are each alkyl, preferably aC₁-C₆ alkyl. In certain embodiments, R¹² and R¹³ are each methyl. Incertain embodiments, R¹¹ and R¹⁴ are each independently alkyl,substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certainembodiments, R¹¹ and R¹⁴ are each independently a C₁-C₆ alkyl, a C₁-C₆substituted alkyl, or sulfoalkyl, which can be the same or different. Incertain embodiments, R_(x) is succinimidyl ester (SE),sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP) ester,tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a method for tracking cell proliferation,differentiation, and/or function, the method being compatible for usewith, for example, flow cytometry and fluorescence microscopy, andcomprising:

a) incubating a mixture of cells and a compound of structural formula(II):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl,-L-R_(x), or -L-S_(C);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹ and R²²are each independently H, halogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) providing a stimulus to the mixture to elicit a fluorescent signal;and

c) analyzing the stimulated mixture.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R³, R⁴, R⁵ and R⁶ are each independently H or halo. Incertain embodiments, R³, R⁴, R⁵ and R⁶ are independently H, Cl, or F. Incertain embodiments, R⁷ and R⁹ are each independently alkyl, substitutedalkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certain embodiments,R⁷ and R⁹ are each independently methyl, -L-R_(x), -L-S_(c) or(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 3. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a method for tracking cell proliferation,differentiation, and/or function, the method being compatible for usewith, for example, flow cytometry and fluorescence microscopy, andcomprising:

a) incubating a mixture of cells and a compound of structural formula(III):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl,-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³,and R²⁴ are each independently H, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) providing a stimulus to the mixture to elicit a fluorescent signal;and

c) analyzing the stimulated mixture.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R⁷, R⁹, R¹⁷, R¹⁹, R²⁰, R²², R²³ and R²⁴ are eachindependently alkyl, substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or-L-S_(c). In certain embodiments, R⁷, R⁹, R¹⁷, R²², R²³ and R²⁴ are eachmethyl. In certain embodiments, R¹⁹ and R²⁰ are each independently(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 1. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a method for tracking cell proliferation,differentiation, and/or function, the method being compatible for usewith, for example, flow cytometry and fluorescence microscopy, andcomprising:

a) incubating a mixture of cells and a compound of structural formula(IV):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl,-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³,and R²⁴ are each independently H, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) providing a stimulus to the mixture to elicit a fluorescent signal;and

c) analyzing the stimulated mixture.

In certain embodiments, R⁹, R¹², R¹³, R²² and R²³ are each alkyl,preferably a C₁-C₆ alkyl. In certain embodiments, R⁹, R¹², R¹³, R²² andR²³ are each methyl. In certain embodiments, R³, R⁴, R⁵ and R⁶ are eachindependently H or halo. In certain embodiments, R³ and R⁶ areindependently H, Cl, or F. In certain embodiments, R² is carboxyl. Incertain embodiments, R⁷, R⁹ and R²⁰ are each independently alkyl,substituted alkyl, sulfoalkyl, sulfo, (CH₂)_(n)SO₃ ⁻, wherein n is aninteger between 1 and 6, -L-R_(x), or -L-S_(c). In certain embodiments,R⁷ is -L-R_(x) and R²⁰ is (CH₂)_(n)SO₃ ⁻. In certain embodiments, nis 1. In certain embodiments, R_(x) is succinimidyl ester (SE),sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP) ester,tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

In certain embodiments, R_(x) is selected from an acrylamide, anactivated ester of a carboxylic acid, a carboxylic ester, an acyl azide,an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline,an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a photoactivatable group, a reactive platinum complex,a silyl halide, a sulfonyl halide, and a thiol. In certain embodimentsthe reactive group is selected from the group consisting of carboxylicacid, succinimidyl ester of a carboxylic acid, hydrazide, amine and amaleimide. The reactive group may be attached to any appropriate site onthe reporter molecule or the aniline moiety. In certain embodiments, atleast one member selected from R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹²,R¹³, R¹⁹ and R²⁰ is a reactive group. In certain embodiments, at leastone of R², R⁴, R⁵, R⁷, R⁸, R¹⁰, R¹² and R¹³ is a reactive group. Incertain embodiments, at least one of R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is areactive group.

In certain embodiments, S_(c) is selected from a carrier molecule and asolid support. In certain embodiments, S_(c) is selected from an aminoacid, a polymer of amino acids, a peptide, a protein, a neurotoxin, aphallotoxin, a cytokine, a toxin, a protease substrate, a protein kinasesubstrate, an enzyme, an antibody, an antibody fragment, a lectin, aglycoprotein, a histone, an albumin, a lipoprotein, avidin,streptavidin, protein A, protein G, a phycobiliprotein, a fluorescentprotein, a hormone, a growth factor, a nucleic acid base, a nucleoside,a nucleotide, a nucleic acid polymer, a nucleotide analog, a nucleosideanalog, a nucleoside triphosphate, a deoxynucleoside triphosphate, adideoxynucleoside triphosphate, a hapten, a carbohydrate, apolysaccharide, a lipid, an ion-complexing moiety (such as a crownether), a PEG group, and an organic or inorganic polymer. In certainembodiments, at least one member selected from R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁹ and R²⁰ is a conjugated substance. In certainembodiments, at least one of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ isa conjugated substance. In certain embodiments, at least one of R⁷, R⁸,R⁹, R¹⁰, R¹² and R¹³ is a conjugated substance. In certain embodiments,at least one of R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴,R¹⁵ and R¹⁶ is a PEG group.

In certain embodiments, the compounds used in the methods providedherein are selected from the group consisting of Compound 1, Compound 2,Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8,Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19,Compound 20, Compound 21, Compound 22, Compound 23, Compound 24,Compound 25 and amine-reactive forms thereof.

Another embodiment provides a method for determining cell health and/orcell viability, the method being compatible for use with, for example,flow cytometry and fluorescence microscopy, and comprising:

a) incubating a mixture of cells and a compound of structural formula(I):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl, or-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹R¹⁰, R¹¹, R¹⁴, R¹⁵, and R¹⁶ are eachindependently H, halogen, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c); or

R⁷ taken together with R¹⁴ are part of an optionally substituted 5- or6-membered ring;

R⁸ taken together with R¹⁵ are part of an optionally substituted 5- or6-membered ring;

R⁹ taken together with R¹¹ are part of an optionally substituted 5- or6-membered ring;

R¹⁰ taken together with R¹⁶ are part of an optionally substituted 5- or6-membered ring;

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) providing a stimulus to the mixture to elicit a fluorescent signal;and

c) analyzing the stimulated mixture.

In certain embodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). Incertain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are each independently H,alkyl, substituted alkyl, or sulfoalkyl. In certain embodiments, R⁷, R⁸,R⁹, and R¹⁰ are each independently a C₁-C₆ alkyl, which can be the sameor different. In certain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are eachmethyl. In certain embodiments, R¹² and R¹³ are each alkyl, preferably aC₁-C₆ alkyl. In certain embodiments, R¹² and R¹³ are each methyl. Incertain embodiments, R¹¹ and R¹⁴ are each independently alkyl,substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certainembodiments, R¹¹ and R¹⁴ are each independently a C₁-C₆ alkyl, a C₁-C₆substituted alkyl, or sulfoalkyl, which can be the same or different. Incertain embodiments, R_(x) is succinimidyl ester (SE),sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP) ester,tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a method for determining cell health and/orcell viability, the method being compatible for use with, for example,flow cytometry and fluorescence microscopy, and comprising:

a) incubating a mixture of cells and a compound of structural formula(II):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl,-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹ and R²²are each independently H, halogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) providing a stimulus to the mixture to elicit a fluorescent signal;and

c) analyzing the stimulated mixture.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R³, R⁴, R⁵ and R⁶ are each independently H or halo. Incertain embodiments, R³, R⁴, R⁵ and R⁶ are independently H, Cl, or F. Incertain embodiments, R⁷ and R⁹ are each independently alkyl, substitutedalkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certain embodiments,R⁷ and R⁹ are each independently methyl, -L-R_(x), -L-S_(c) or(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 3. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a method for determining cell health and/orcell viability, the method being compatible for use with, for example,flow cytometry and fluorescence microscopy, and comprising:

a) incubating a mixture of cells and a compound of structural formula(III):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl,-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³,and R²⁴ are each independently H, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) providing a stimulus to the mixture to elicit a fluorescent signal;and

c) analyzing the stimulated mixture.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R⁷, R⁹, R⁷, R¹⁹, R²⁰, R²², R²³ and R²⁴ are eachindependently alkyl, substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or-L-S_(c). In certain embodiments, R⁷, R⁹, R¹⁷, R²², R²³ and R²⁴ are eachmethyl. In certain embodiments, R¹⁹ and R²⁰ are each independently(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 1. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a method for determining cell health and/orcell viability, the method being compatible for use with, for example,flow cytometry and fluorescence microscopy, and comprising:

a) incubating a mixture of cells and a compound of structural formula(IV):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl,-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³,and R²⁴ are each independently H, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) providing a stimulus to the mixture to elicit a fluorescent signal;and

c) analyzing the stimulated mixture.

In certain embodiments, R⁹, R¹², R¹³, R²² and R²³ are each alkyl,preferably a C₁-C₆ alkyl. In certain embodiments, R⁹, R¹², R¹³, R²² andR²³ are each methyl. In certain embodiments, R³, R⁴, R⁵ and R⁶ are eachindependently H or halo. In certain embodiments, R³ and R⁶ areindependently H, Cl, or F. In certain embodiments, R² is carboxyl. Incertain embodiments, R⁷, R⁹ and R²⁰ are each independently alkyl,substituted alkyl, sulfoalkyl, sulfo, (CH₂)_(n)SO₃ ⁻, wherein n is aninteger between 1 and 6, -L-R_(x), or -L-S_(c). In certain embodiments,R⁷ is -L-R_(x) and R²⁰ is (CH₂)_(n)SO₃ ⁻. In certain embodiments, nis 1. In certain embodiments, R_(x) is succinimidyl ester (SE),sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP) ester,tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

In certain embodiments, R_(x) is selected from an acrylamide, anactivated ester of a carboxylic acid, a carboxylic ester, an acyl azide,an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline,an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a photoactivatable group, a reactive platinum complex,a silyl halide, a sulfonyl halide, and a thiol. In certain embodimentsthe reactive group is selected from the group consisting of carboxylicacid, succinimidyl ester of a carboxylic acid, hydrazide, amine and amaleimide. The reactive group may be attached to any appropriate site onthe reporter molecule or the aniline moiety. In certain embodiments, atleast one member selected from R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹²,R¹³, R¹⁹ and R²⁰ is a reactive group. In certain embodiments, at leastone of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is a reactive group. Incertain embodiments, at least one of R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is areactive group.

In certain embodiments, S_(c) is selected from a carrier molecule and asolid support. In certain embodiments, S_(c) is selected from an aminoacid, a polymer of amino acids, a peptide, a protein, a neurotoxin, aphallotoxin, a cytokine, a toxin, a protease substrate, a protein kinasesubstrate, an enzyme, an antibody, an antibody fragment, a lectin, aglycoprotein, a histone, an albumin, a lipoprotein, avidin,streptavidin, protein A, protein G, a phycobiliprotein, a fluorescentprotein, a hormone, a growth factor, a nucleic acid base, a nucleoside,a nucleotide, a nucleic acid polymer, a nucleotide analog, a nucleosideanalog, a nucleoside triphosphate, a deoxynucleoside triphosphate, adideoxynucleoside triphosphate, a hapten, a carbohydrate, apolysaccharide, a lipid, an ion-complexing moiety (such as a crownether), a PEG group, and an organic or inorganic polymer. In certainembodiments, at least one member selected from R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁹ and R²⁰ is a conjugated substance. In certainembodiments, at least one of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ isa conjugated substance. In certain embodiments, at least one of R⁷, R⁸,R⁹, R¹⁰, R¹² and R¹³ is a conjugated substance. In certain embodiments,at least one of R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴,R¹⁵ and R¹⁶ is a PEG group.

In certain embodiments, the compounds used in the methods providedherein are selected from the group consisting of Compound 1, Compound 2,Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8,Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19,Compound 20, Compound 21, Compound 22, Compound 23, Compound 24,Compound 25 and amine-reactive forms thereof.

In one illustrative embodiment, the method includes a second compoundexcitable at a different wavelength as the first compound. In anotherillustrative embodiment, the method includes a second compound where thesecond compound is selected from FAM-conjugated IgG or GFP.

In another illustrative embodiment of the method, step a) is conductedfor approximately 20 minutes. In another illustrative embodiment, stepb) and step c) are carried out concurrently. In another illustrativeembodiment, step b) and step c) involve flow cytometry.

In addition to the methods described herein for tracking cellproliferation, differentiation, and/or function, the present disclosurealso provides methods of using the compounds, including conjugatesthereof, described herein to detect an analyte in a sample. Those ofskill in the art will appreciate that this focus is for clarity ofillustration and does not limit the scope of the methods in which thecompounds disclosed herein find use.

The compounds provided herein can be used to stain biological samples,i.e. samples that comprise biological components. In one embodiment, thesample comprises heterogeneous mixtures of components, including intactcells, cell extracts, bacteria, viruses, organelles, and mixturesthereof. In another aspect, the sample comprises a single component orhomogeneous group of components, e.g. biological polymers such as aminoacid polymers, nucleic acid polymers or carbohydrate polymers, or lipidmembrane complexes, whether the polymers are synthetic or natural.

The sample is typically stained by passive means, i.e., by incubationwith a solution of a compound provided herein. Any other method ofintroducing the compound into the sample, such as microinjection of asolution containing the compound into a cell or organelle, can be usedto accelerate introduction of the compound into the sample. Thecompounds provided herein are generally non-toxic to living cells andother biological components, within the concentrations of use.

In certain embodiments, the compounds disclosed herein are utilized tostain a sample to give a detectable optical response under desiredconditions by a) preparing a dye solution comprising a compounddescribed above, at a concentration sufficient to yield a detectableoptical response under the desired conditions; combining the sample ofinterest with the dye solution for a period of time sufficient for thecompound to yield a detectable optical response under the desiredconditions; and c) illuminating the sample at a wavelength selected toelicit the optical response. Optionally, the sample is washed to removeresidual, excess or unbound compound. The compound typically forms acovalent or non-covalent association or complex with an element of thesample, or is simply present within the bounds of the sample or portionof the sample.

The sample can be observed immediately after staining. The sample isoptionally combined with other solutions in the course of staining,including wash solutions, permeabilization and/or fixation solutions,and other solutions containing additional detection reagents. Washingfollowing staining generally improves the detection of the opticalresponse due to the decrease in non-specific background fluorescenceafter washing. Satisfactory visualization is possible without washing byusing lower labeling concentrations. A number of fixatives and fixationconditions suitable for practicing this invention are known in the art,including formaldehyde, paraformaldehyde, formalin, glutaraldehyde, coldmethanol and 3:1 methanol:acetic acid. Fixation is typically used topreserve cellular morphology and to reduce biohazards when working withpathogenic samples. Selected embodiments of the compounds describedabove are well retained in cells, and sample cells stained with thesecompounds retain considerable fluorescent staining after fixation.Fixation is optionally followed or accompanied by permeabilization, suchas with acetone, ethanol, DMSO or various detergents, to allow bulkycompounds, including conjugates described above, to cross cellmembranes, according to methods generally known in the art. The stainingof the present disclosure is optionally combined with the use of anadditional detection reagent that produces a detectable response due tothe presence of a specific cell component, intracellular substance, orcellular condition, according to methods generally known in the art.Where the additional detection reagent has spectral properties thatdiffer from those of the subject compounds, multi-color applications arepossible.

The compounds provided herein are also of use to derivative lowmolecular weight compounds for their analysis by capillary zoneelectrophoresis (CZE), HPLC or other separation techniques.

In one embodiment, the staining is used to determine a specifiedcharacteristic of the sample by further comparing the optical responsewith a standard or expected response. For example, the dye solution isused to monitor specific components of the sample with respect to theirspatial and temporal distribution in the sample. Alternatively, the dyepreferentially binds to a specific analyte in a sample, enabling theresearcher to determine the presence or quantity of that specificanalyte. In another embodiment, the dye solution is used to analyze thesample for the presence of a mechanism that responds specifically to thecompound, such as oxidation or reduction. The desired analysis to beperformed determines the composition of the dye solution and chemicalnature of the compound itself. In another example, the compound is boundby an antibody directed against the compound, typically resulting in thefluorescence quenching of the compound.

For biological applications, the dye solution is typically an aqueous ormostly aqueous solution that comprises one or more of the describedcompounds. In one aspect of the disclosure, the dye solution comprises acompound as described above; alternatively, the dye solution comprises acompound that is a reactive derivative of the compound, as previouslydescribed.

In yet another exemplary embodiment, the dye solution includes aconjugate of the compound as described above.

In yet another embodiment, a composition that includes a first conjugateof a compound described herein in combination with a second conjugate ofa compound provided herein. The second conjugate includes a componentthat is covalently bonded to a second fluorophore. The first and secondfluorophore have different structures and preferably fluoresce atdifferent wavelengths. Even more preferably, the first and secondfluorophores are selected so that their fluorescence emissionsessentially do not overlap.

The fluorophore on the second conjugate can include substantially anyfluorescent structure known in the art including, but not limited to,small organic fluorophores, fluorescent proteins, and reporter groupsthat are not necessarily fluorescent but which, under correctconditions, convert a fluorogenic substrate into a fluorophore, e.g.,horseradish peroxidase. Exemplary second fluorophores of use hereininclude those that include a moiety that is a member selected from acoumarin, a xanthene (e.g., fluorescein), a cyanine, a pyrene, aborapolyazaindacene, an oxazine, and bimane.

The components of the mixture may be the same or a different molecule.The discussion herein pertaining to the identity of various componentsis generally applicable to this embodiment of the present disclosure.

In another exemplary embodiment, at least one of the first and secondconjugates is bound to a molecule for which it is a binding partner.

The present disclosure also provides a method for detecting an analytein a sample. The method includes contacting the sample with a conjugateof a compound provided herein in which the component is a bindingpartner for the analyte. The mixture of the conjugate and the analyte isincubated under any appropriate conditions for a length of timesufficient for at least a fraction of the analyte population to interactwith the conjugate. The interaction can be by any known interactionmechanism, and the present disclosure is not limited to application withany single type of analyte-conjugate interaction mechanism. Theinteraction between the analyte and the compound conjugate results inthe formation of a fluorescent analyte. The fluorescent analyte isreadily detected and/or quantitated by irradiating it with light of awavelength that causes the fluorescent analyte to emit fluorescence.

In the method described above, any number or combination ofpurification, separation or derivatization steps are optionally includedas steps in the method. In an exemplary embodiment, the fluorescentanalyte is separated from the remainder of the sample, fromnon-fluorescent analyte or from excess unbound conjugate prior todetermining the fluorescence of the fluorescent analyte.

In another exemplary embodiment, the present disclosure provides amulticolor method for detecting an analyte or more than one analyte. Forexample, when it is desired to detect, and particularly to confirm theidentity of an analyte, more than one fluorescent conjugate, preferablyfluorescing at different wavelengths can be co-localized on the singleanalyte.

The use of more than one color of fluorescent conjugate per analyteprovides assays in which specificity is dramatically increased, byrequiring that the different colors or color combinations of thefluorescent conjugates coincide spatially during detection. This candramatically reduce or even eliminate the detection of nonspecificallybound targets or labels, enhancing specificity and sensitivity of theassay. Underlying the improvement represented by the use of multipledifferently colored fluorescent conjugates is the improbability ofaccidentally encountering two or more preselected different colors atthe same location at the same time. The improbability increases as morefluorescent conjugates of different colors are used. Alternatively, inanother exemplary embodiment, the emission from the two or moredifferently colored fluorescent conjugates combines to form a thirdcolor, which is not otherwise present in the assay.

In an exemplary application of the present method, different features ofan analyte, e.g., a cell or epitopes of a molecule, are labeled withdifferent colored fluorescent conjugates. The target is detected and itsidentity is confirmed using the colocalization or “coincidence” of eachcolor on each target. Coincidence staining allows for the detection anddifferentiation of different organisms or strains of organismsexpressing different surface markers. Moreover, coincidence stainingprovides a method of distinguishing molecules of different structuredown to the level of isomeric differences and differences instereochemistry.

In the detection of pathogenesis, the most direct analyte is thepathogenic organism itself. In this case, assays preferably identifyparticular features of the organism such as surface proteins. To furtheraid in characterization, it is preferred to assay for molecular analytesas well. An example of a molecular analyte is an exotoxin such ascholera toxin. Antigen specific binding receptors are generated thatrecognize different characteristics of an analyte with high specificity.In the case of molecular analytes, receptors recognize differentepitopes of a protein or small molecule, while cellular analytes arerecognized through different molecules on the cell surface.

Although the fluorescence from each conjugate can be detectedsimultaneously, in one embodiment, to facilitate coincidence staining,the fluorescence from each analyte is detected independently.

In another exemplary embodiment, colocalization is used to differentiatebetween the formation of an analyte-conjugate complex and non-specificbinding of the analyte to another species within the assay system. Theintrinsic sensitivity of an assay often is limited by non-specificbinding of the analyte or other assay mixture components to thesubstrate. Single analyte coincidence staining can be used todifferentiate between specific binding of the analyte to the conjugateand nonspecific binding of assay mixture components to the conjugatebased on the colocalization of fluorescent conjugate colors. Those ofskill in the art will appreciate that coincidence staining as describedherein is useful to distinguish non-specific binding in both solid-phase(e.g., gene chip) and solution-based assays

Coincidence staining can also be used to identify a single analyte. Forexample, one may wish to confirm the presence of a selected analyte in amixture of analytes that are structurally similar (e.g. having a commonepitope) or that have similar affinity for the component of theconjugate. In such circumstances, it may prove that the detection of asingle epitope is not sufficient for conclusive identification of atarget. Measuring the level of 2, preferably 3, more preferably 4 andeven more preferably 5 or more markers within a single analyte, providesan unambiguous profile specific for the analyte of interest.

In another exemplary embodiment, the present disclosure provides amethod for distinguishing between organisms expressing the same surfacemarkers. Using coincidence staining, it is possible to identifydifferences in targets based on the ratio of surface marker expression.For example, despite intense efforts, no single binding-receptor hasbeen found for the unambiguous detection of B. anthracis spores, due toextensive cross-reactivity with related B. cereus and B. thuringiensis,which are genetically a single species (Helgason et al., Appl. Envir.Microbiol. 66:2627-2630 (2000)). Despite being of the same species,however, the relative amount of various surface proteins is differentbetween the three bacilli. Tus, multipoint detection of a variety ofmarkers at the single cell level will provide the specificity requiredto detect B. anthracis.

Diagnostic tests that detect the presence or absence of a single markerare unable to distinguish among strains that are nearly identical at thegenetic level, highlighting the need for new tools to distinguishbetween closely related organisms. Epidemics caused by emerging variantsof known pathogens are a common theme in infectious diseases (Jiang etal., Appl Environ Microbiol 66: 148-53 (2000)) (Hedelberg et al. Nature406:477-483 (2000)). There is also the problem of deliberate engineeringof pathogens, incorporating virulence determinants from other species.An attack by such pathogens would be misdiagnosed due to the presence ofmarkers not normally found in the attacking pathogen. By probingmultiple markers within a single organism, using the methods providedherein, such variants are detected and preferably identified.

Detection by eye is also useful in those embodiments that rely oncoincidence staining. The human eye is extremely good at distinguishingbetween subtly different combinations of colors, especially when thecolors are chosen correctly. By way of illustration, it is trivial forpeople to distinguish between the colors red, green and yellow. Yellow,however, is simply the spectral sum of red and green, so if red andgreen fluorescent conjugates are used for coincidence staining, positiveassay signal can easily be identified by the perceived color, yellow.Other color combinations of use in this embodiment of the disclosurewill be readily apparent to those of skill in the art, such ascombinations of red, green and blue to form white.

In another exemplary embodiment, the present disclosure provides amethod for detecting a first analyte and a second analyte in a sample.The method includes incubating the sample with a compound providedherein that includes first and second fluorescent labeled conjugates.The component of the first conjugate is a binding partner for the firstanalyte and the component of the second conjugate is a binding partnerfor the second analyte. The incubation continues for a time and underconditions appropriate to induce an interaction between at least afraction of the population of the first analyte with the firstconjugate. During this incubation period, it is generally preferred thata similar interaction occurs between the second analyte and secondconjugate, however, it is within the scope of the present disclosure tochange the incubation conditions as necessary to drive the formation ofa conjugate-analyte complex between the second conjugate and secondanalyte.

Following the formation of at least the first analyte-conjugate complex,the sample is illuminated with light of a wavelength appropriate tocause the complex to fluoresce, thereby detecting the first analyte. Thesecond analyte is detected in a similar manner and may be detectedsimultaneously with the first analyte or by the sequential illuminationof the sample with wavelengths appropriate to induce the fluorescence ofeach fluorescent conjugate.

Solutions of the compounds provided herein are prepared according tomethods generally known in the art. As with related known fluorophoresand fluorogens, the compounds and conjugates of the compounds providedherein are generally soluble in water and aqueous solutions having a pHgreater than or equal to about 6. Stock solutions of pure dyes, however,are typically dissolved in organic solvent before diluting into aqueoussolution or buffer. Preferred organic solvents are aprotic polarsolvents such as DMSO, DMF, N-methylpyrrolidone, acetone, acetonitrile,dioxane, tetrahydrofuran and other nonhydroxylic, completelywater-miscible solvents. In general, the amount of the compound orconjugate of the compound in the dye solution is the minimum amountrequired to yield detectable staining in the sample within a reasonabletime, with minimal background fluorescence or undesirable staining. Theexact concentration of the compound or conjugate of the compound to beused is dependent upon the experimental conditions and the desiredresults, and optimization of experimental conditions is typicallyrequired to determine the best concentration of stain to be used in agiven application. The concentration of the compound present in the dyesolution typically ranges from nanomolar to micromolar. The requiredconcentration for the dye solution is determined by systematic variationin the concentration of the compound or conjugate of the compound untilsatisfactory staining is accomplished. The starting ranges are readilydetermined from methods known in the art for use of similar compoundsunder comparable conditions for the desired optical response.

For those compounds of the present disclosure that are lipophilic innature, the compound is optionally introduced into living cells bypassive permeation through the cellular membranes. Less cell-permeantcompounds provided herein can be introduced into cells by pressuremicroinjection methods, scrape loading techniques (short mechanicaldisruption of the plasma membrane where the plasma membrane is peeledaway from the cytoplasm, the compound is perfused through the sample andthe plasma membrane is reassembled), patch clamp methods (where anopening is maintained in the plasma membrane for long periods) orphagocytosis. Any other treatment that will permeabilize the plasmamembrane, such as electroporation, shock treatments or highextracellular ATP can be used to introduce the compound, conjugate ofthe compound, or blocked compound into the cellular cytoplasm.

In an exemplary embodiment, the dye solution comprises a compound thatnon-covalently associates with organic or inorganic materials. Exemplaryembodiments of the compounds that possess a lipophilic substituent canbe used to stain lipid assemblies such as biological membranes orliposomes by non-covalent incorporation of the compound within themembrane, e.g. for use as probes for membrane structure or forincorporation in liposomes, lipoproteins, films, plastics, lipophilicmicrospheres or similar materials.

The compounds provided herein are useful as coloring agents, tracers fordetecting the flow of fluids such as in angiography, and tracing offluid flow through gap junctions of neurons according to proceduresknown in the art for other dyes. The compounds provided herein are alsouseful in assays as haptens, according to known methods, wherein thecompound is recognized by an anti-compound antibody.

Reactive versions of the compounds provided herein can be used to labelcell surfaces, cell membranes or intracellular compartments such asorganelles, or in the cell's cytoplasm. Certain reactive groups allowthe retention of the compound in cells or organelles by reacting withcellular materials. In particular, haloalkyl- orhalomethylbenzamide-substituted fluorophores are used to reactselectively with intracellular components such as glutathione, or toretain the compounds within cells or within selected organelles wherethe compound is localized therein, according to methods previouslydescribed (U.S. Pat. No. 5,362,628 to Haugland et al, (1994); U.S. Pat.No. 5,576,424 to Mao et al. (1996) (in cells); and U.S. Pat. No.5,459,268 to Haugland et al. (1995) and U.S. Pat. No. 5,686,261 to Zhanget al. (1997) (in mitochondria); all incorporated by reference).Polyfluoroaryl-substituted compounds are similarly retained in cells, inpart by covalent attachment. The reactive compounds are used to localizestaining in a part of the sample, e.g., where the localization of thecorresponding functional group is indicative of a characteristic of thesample; or to retain the compound in a specific portion of the samplefor extended periods of time, e.g., to follow the stained portion of thesample through a period of time or sequence of events. Alternatively,the compounds are used according to this method to make conjugates, asdescribed above, which are separately useful for staining.

In an exemplary embodiment in which the dye solution comprises aconjugate of the compound, the conjugate is a labeled member of aspecific binding pair, and is used as a fluorescent probe for thecomplementary member of that specific binding pair, each specificbinding pair member having an area on the surface or in a cavity whichspecifically binds to and is complementary with a particular spatial andpolar organization of the other. The fluorescent conjugate of a specificbinding pair member is useful for detecting and optionally quantifyingthe presence of the complementary specific binding pair member in asample, by methods that are well known in the art. Optionally, thecomplementary binding pair member is present in an animal cell, plantcell, bacteria, yeast or virus. Alternatively, the complementary memberis immobilized on a solid or semi-solid surface, such as a polymer,polymeric membrane or polymeric particle (such as a polymeric bead). Theconjugate of the compound may also comprise a compound in a blocked formwherein the block is later removed by the action of an enzyme or light.

Representative specific binding pairs are shown in Table 2. Typically aspecific binding pair member conjugated to the compound is a ligand or areceptor. As used herein, the term ligand means any organic compound forwhich a receptor naturally exists or can be prepared. A receptor is anycompound or composition capable of recognizing a spatial or polarorganization of a molecule, e.g. epitopic or determinant site. Ligandsfor which naturally occurring receptors exist include natural andsynthetic proteins, including avidin and streptavidin, antibodies,enzymes, and hormones; nucleotides and natural or syntheticoligonucleotides, including primers for RNA and single- anddouble-stranded DNA; lipids; polysaccharides and carbohydrates; and avariety of drugs, including therapeutic drugs and drugs of abuse andpesticides. The compounds provided herein are used according to methodsextensively known in the art, to prepare antibody conjugates for use inmicroscopy and immunofluorescent assays and nucleotide oroligonucleotide conjugates for nucleic acid hybridization assays andnucleic acid sequencing (e.g., U.S. Pat. No. 5,332,666 to Prober, et al.(1994); U.S. Pat. No. 5,171,534 to Smith, et al. (1992); U.S. Pat. No.4,997,928 to Hobbs (1991); and PCT International Application PublicationNo. WO 94/05688 to Menchen, et al.) and a wide variety of otherapplications. Nucleotide conjugates are readily incorporated by DNApolymerase and can be used for in situ hybridization or othertechniques.

In another preferred embodiment, the compounds described herein areutilized as a component of one or more probes used in a multiplex assayfor detecting one or more species in a mixture. As used herein, the term“multiplex assay” refers to an assay in which fluorescence from two ormore dyes is detected, or in which fluorescence energy transfer betweentwo or more dyes and one or more quencher is detected.

Probes that include a compound of the present disclosure areparticularly useful in performing multiplex-type analyses and assays. Ina typical multiplex analysis, two or more distinct species (or regionsof one or more species) are detected using two or more probes, whereineach of the probes is labeled with a different fluorophore. Preferredspecies used in multiplex analyses generally meet at least two criteria:the fluorescent species is bright and spectrally well resolved; and thebackground fluorescence of the first dye does not significantly overlapthe emission range of the second dye.

Thus, in a further embodiment, the present disclosure provides a mixturecomprising at least a first and a second compound provided herein. Thefirst and second compounds are preferably conjugated to a component of aconjugate. The compounds may be conjugated to the same component or todifferent components.

The compounds provided herein allow for the design of multiplex assaysin which more than one compound is used in the assay. A number ofdifferent multiplex assays using the compounds provided herein will beapparent to one of skill in the art. In one exemplary assay, each of theat least two distinct dyes are detected. Alternatively, an assay can bepracticed in which each distinct compound transfers energy to a distinctquencher to which the compound is “matched.” The fluorophores can bebound to the same molecule as the quencher or to a different molecule.Moreover, similar to the dyes and the quenchers, the component of thedifferent conjugates of use in a particular assay system can be the sameor different.

In addition to the mixtures described above, the present disclosure alsoprovides a method for detecting or quantifying a particular molecularspecies. The method includes: (a) contacting the species with a mixturesuch as that described above; and (b) detecting a change in afluorescent property of one or more component of the mixture, themolecular species or a combination thereof, thereby detecting orquantifying the molecular species.

The simultaneous use of two or more probes using donor-acceptor energytransfer is known in the art. For example, multiplex assays usingnucleic acid probes with different sequence specificities have beendescribed. Fluorescent probes have been used to determine whether anindividual is homozygous wild-type, homozygous mutant or heterozygousfor a particular mutation. For example, using one quenched-fluoresceinmolecular beacon that recognizes the wild-type sequence and anotherrhodamine-quenched molecular beacon that recognizes a mutant allele, itis possible to genotype individuals for the β-chemokine receptor(Kostrikis et al. Science 279:1228-1229 (1998)). The presence of only afluorescein signal indicates that the individual is wild-type, and thepresence of rhodamine signal only indicates that the individual is ahomozygous mutant. The presence of both the rhodamine and fluoresceinsignal is diagnostic of a heterozygote. Tyagi et al. NatureBiotechnology 16: 49-53 (1998)) have described the simultaneous use offour differently labeled molecular beacons for allele discrimination,and Lee et al., BioTechniques 27: 342-349 (1999) have described sevencolor homogenous detection of six PCR products.

The compounds provided herein can be used in multiplex assays designedto detect and/or quantify substantially any species, including, forexample, whole cells, viruses, proteins (e.g., enzymes, antibodies,receptors), glycoproteins, lipoproteins, subcellular particles,organisms (e.g., Salmonella), nucleic acids (e.g., DNA, RNA, andanalogues thereof), polysaccharides, lipopolysaccharides, lipids, fattyacids, non-biological polymers and small molecules (e.g., toxins, drugs,pesticides, metabolites, hormones, alkaloids, steroids).

The compounds of the present disclosure are also of use in the numerousfluorescence polarization assays that use conjugates of fluorescent dyesto low molecular weight drugs and ligands, which will be improved by theuse of the dye compounds of the invention, e.g., U.S. Pat. No. 4,420,568to Wang (1983) and U.S. Pat. No. 4,510,251 to Kirkemo et al. (1985).

In those embodiments in which a compound is conjugated to a specificbinding pair member that is a chelator of calcium, sodium, magnesium,potassium, or other biologically important metal ion, the conjugate ofthe compound functions as an indicator of the ion, which indicators areoptionally further conjugated to a biological or plastic polymeraccording to methods known in the art; e.g., using analogs of thecompounds described in U.S. Pat. No. 5,453,517 to Kuhn, et al. (1995);U.S. Pat. No. 5,405,975 to Kuhn, et al. (1995). Alternatively, thecompound itself acts as a pH indicator at pH values within about 1.5 pHunits of the individual compound's pKa. Typically the detectable opticalresponse of the ion indicators is a change in fluorescence.

In another exemplary embodiment, the compounds provided herein aresubstrates for enzymes, such as caspases, oxidases and other reactiveoxidizing agents, such as peroxidase enzymes.

Polypeptides may be conjugated, or “labeled”, with labeling reagents toprepare the peptide conjugates provided herein. Peptides, proteins,antibodies, and other biopolymers comprised of amino acids and aminoacid analogs may be covalently labeled by conjugation with the compoundsprovided herein. Typically, the compounds bear an electrophilic linkingmoiety which reacts with a nucleophilic group on the peptide, e.g. aminoterminus, or side-chain nucleophile of an amino acid. Alternatively, thecompound may be in nucleophilic form, e.g. amino- or thiol-linkingmoiety, which reacts with an electrophilic group on the peptide, e.g.NHS of the carboxyl terminus or carboxyl side-chain of an amino acid.The polypeptide may be on a solid support, i.e. synthesis resin, duringthe labeling reaction. Alternatively, the polypeptide may have beencleaved prior to labeling. By appropriate selection of protectinggroups, certain reactive functionality on the peptide can be selectivelyunmasked for reaction with a labeling reagent.

The compound label reagents include a reactive linking group, “linkingmoiety”, at one of the substituent positions for covalent attachment ofthe compound to a polypeptide. Linking moieties capable of forming acovalent bond are typically electrophilic functional groups capable ofreacting with nucleophilic molecules, such as alcohols, alkoxides,amines, hydroxylamines, and thiols. Examples of electrophilic linkingmoieties include succinimidyl ester, isothiocyanate, sulfonyl chloride,sulfonate ester, silyl halide, 2,6-dichlorotriazinyl, pentafluorophenylester, phosphoramidite, maleimide, iodoacetamide, haloacetyl, epoxide,alkylhalide, allyl halide, aldehyde, ketone, acylazide, and anhydride.

One linking moiety is an N-hydroxysuccinimidyl ester (NHS) of a carboxylgroup substituent on a compound provided herein. The NHS ester form ofthe compound is an exemplary labeling reagent. Another preferred linkingmoiety is a phosphoramidite reagent of the dyes provided herein.Phosphoramidite dye reagents are particularly useful for labeling ofpolypeptides by automated synthesis on solid support. Oligonucleotidesare commonly synthesized on solid supports by the phosphoramidite method(Canuthers, U.S. Pat. No. 4,415,732; Caruthers, U.S. Pat. No. 4,458,066;Beaucage, Tetrahedron 48:2223-2311 ((1992)).

Peptide conjugates of the present disclosure are labeled with a compoundprovided herein. The compound provides a detection element forlocalizing, visualizing, and quantitating the cleavage event. Theproperties of the compound also facilitate transport through the cellmembrane and targeting of intracellular structures and molecules. Uponcleavage of the peptide by a peptidase or protease, such as a caspase, adetectable increase in fluorescence from the compound may be measured.The peptide conjugates provided herein retain the specific binding andrecognition properties of the respective compound and peptide sequence.

For example, in Scheme 1 a compound of the present disclosure that isbound to a peptide(s) is non-fluorescent; however, upon cleavage of therecognition site by a protease results in fluorescence of the releaseddye compound.

Certain polypeptide sequences of peptide conjugates are substrates forcaspase enzymes. A protease binding site is an amino acid sequence(polypeptide) which is recognized and cleaved by a particular protease.Caspases are known to cleave polypeptide substrates adjacent toparticular amino acids within a recognition site. A particular caspasedoes not cleave every bond in a substrate that has any particular aminoacid. Rather, caspases are specific to particular amino acid sequenceswhich serve as recognition domains for each particular caspase. Anypolypeptide that comprises the DEVD (SEQ ID NO: 1) caspase recognitionsite can be a peptide conjugate of the present disclosure. In thisrecognition site, the cleavage site is the amide bond between theaspartic acid residue toward the carboxyl terminus and the adjacentamine group. (For example, see Scheme 2).

The enzyme substrates optionally contain additional substituents thatprovide additional advantages. For example, fluorophores modified tocontain a lipophilic tail according to the synthesis described in U.S.Pat. No. 5,208,148 to Haugland et al. (1993), are useful forpermeabilizing substrates for intracellular enzymes.

In another exemplary embodiment, the compounds provided herein are usedto determine the efficiency of a cellular efflux pump of cells in asample. Preferably the compounds are diacetates or diphosphates. Thecompound is used in the minimum concentration that gives a detectablefluorescence emission. Once the diacetate compounds are inside the cell,the blocking acetates are cleaved and the compound becomes highlyfluorescent. The efficiency of the cellular efflux pump of cells in thesample is determined by comparing the fluorescence emission of cells inthe sample with the fluorescence of cells having known effluxefficiency. Where the efflux pump is impaired, inhibited, or absent, thefluorescent compound is well retained in the cell; where the efflux pumpis present and functioning, the fluorescence of the cells decreasesmarkedly. The photostability of the present compounds is advantageousfor monitoring the time course of fluorescence.

Illumination

At any time after or during an assay or staining procedure, the sampleis illuminated with a wavelength of light that results in a detectableoptical response, and observed with a means for detecting the opticalresponse. While the compounds are detectable colorimetrically, usingambient light, typically the compounds are detected by the fluorescenceproperties of the parent fluorophore. Upon illumination, such as by anultraviolet or visible wavelength emission lamp, an arc lamp, a laser,or even sunlight or ordinary room light, the compounds, includingcompounds bound to the complementary specific binding pair member,display intense visible absorption as well as fluorescence emission.Selected equipment that is useful for illuminating the conjugates of thepresent disclosure includes, but is not limited to, hand-heldultraviolet lamps, mercury arc lamps, xenon lamps, argon lasers, laserdiodes, and YAG lasers. These illumination sources are optionallyintegrated into laser scanners, fluorescence microplate readers,standard or mini fluorometers, or chromatographic detectors. Thiscolorimetric absorbance or fluorescence emission is optionally detectedby visual inspection, or by use of any of the following devices: CCDcameras, video cameras, photographic film, laser scanning devices,fluorometers, photodiodes, quantum counters, epifluorescencemicroscopes, scanning microscopes, flow cytometers, fluorescencemicroplate readers, or by means for amplifying the signal such asphotomultiplier tubes. Where the sample is examined using a flowcytometer, a fluorescence microscope or a fluorometer, the instrument isoptionally used to distinguish and discriminate between the compound anda second fluorophore with detectably different optical properties,typically by distinguishing the fluorescence response of the conjugatefrom that of the second fluorophore. Where the sample is examined usinga flow cytometer, examination of the sample optionally includesisolation of particles within the sample based on the fluorescenceresponse of the dye compound by using a sorting device.

A detectable optical response means a change in, or occurrence of, aparameter in a test system that is capable of being perceived, either bydirect observation or instrumentally. Such detectable responses includethe change in, or appearance of, color, fluorescence, reflectance,chemiluminescence, light polarization, light scattering, or x-rayscattering. Typically the detectable response is a change influorescence, such as a change in the intensity, excitation or emissionwavelength distribution of fluorescence, fluorescence lifetime,fluorescence polarization, or a combination thereof. The detectableoptical response may occur throughout the sample or in a localizedportion of the sample. The presence or absence of the optical responseafter the elapsed time is indicative of one or more characteristic ofthe sample. Comparison of the degree of staining with a standard orexpected response can be used to determine whether and to what degreethe sample possesses a given characteristic.

Sample Preparation

The end user will determine the choice of the sample and the way inwhich the sample is prepared. The sample includes, without limitation,any biological derived material or aqueous solution. The sample can be abiological fluid such as whole blood, plasma, serum, nasal secretions,sputum, saliva, urine, sweat, transdermal exudates, cerebrospinal fluid,or the like. Biological fluids also include tissue and cell culturemedium wherein an analyte of interest has been secreted into the medium.Alternatively, the sample may be whole organs, tissue or cells from theanimal. Examples of sources of such samples include muscle, eye, skin,gonads, lymph nodes, heart, brain, lung, liver, kidney, spleen, thymus,pancreas, solid tumors, macrophages, mammary glands, mesothelium, andthe like. Cells include without limitation prokaryotic cells andeukaryotic cells that include primary cultures and immortalized celllines. Eukaryotic cells include without limitation ovary cells,epithelial cells, circulating immune cells, p cells, hepatocytes, andneurons.

When the compounds provided herein are used as components of an assaysystem for a target species in a mixture, such as an enzyme, the targetconcentration is conveniently in the range of about 1 nM to 500 nM, moreusually in the range of about 25 to 250 nM. One may use an individualcompound provided herein, multiple compounds provided herein or acombination of a compound provided herein and a fluorophore or quencherof a different structure in order to detect the presence of or determinethe characteristics of a target in a sample.

When the components of the present disclosure are species that bind totargets that are specific biological structures (e.g., enzymes,receptors, ligands, antigens, antibodies, etc.), the reaction timebetween the compound or conjugate provided herein and the target willusually be at least about 5 min, more usually at least about 30 min andpreferably not more than about 180 min, preferably not more than about120 min, depending upon the temperature, concentrations of enzyme andsubstrate, etc. By using a specific time period for the reaction ortaking aliquots at 2 different times, the rate of reaction can bedetermined for comparison with other determinations. The temperaturewill generally be in the range of about 20 to 50° C., more usually inthe range of about 25 to 40° C.

If the assay focuses on an enzyme, coenzyme, if any, is preferablypresent in excess, so as not be rate limiting. Generally, with theconcentrations of enzyme indicated above, the concentration of coenzymewill be at least about 0.1 mM, usually at least about 1 mM and not morethan about 25 mM. The coenzyme solution should be prepared freshly foreach series of determinations.

Various buffers can be used in the assays provided herein. These buffersinclude PBS, Tris, MOPS, HEPES, phosphate, etc. The pH will varydepending upon the particular assay system, generally within a readilydeterminable range wherein one or more of the sulfonic acid moieties isdeprotonated. The concentration of buffer is generally in the range ofabout 0.1 to 50 mM, more usually 0.5 to 20 mM.

In certain instances, it may be advantageous to add a small amount of anon-ionic detergent to the sample. Generally the detergent will bepresent in from about 0.01 to 0.1 vol. %. Illustrative non-ionicdetergents include the polyoxyalkylene diols, e.g. PLURONICS (BASF),TWEENs (Croda International, PLC), TRITON X-100 (Dow Pharmaceuticals),etc.

After sufficient time for a detectable amount of product to form, thereaction is optionally quenched. Various quenching agents may be used,both physical and chemical. Conveniently, a small amount of awater-soluble inhibitor may be added, such as acetonitrile, DMSO, SDS,methanol, DMF, etc. The amount of inhibitor will vary with the nature ofthe inhibitor and may be determined empirically.

Kits:

Another embodiment provides a kit for tracking cell proliferation,differentiation and/or function, the kit being compatible for use with,for example, flow cytometry and fluorescence microscopy, and comprising:

a) a compound of structural formula (I):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl,-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵, and R¹⁶ are eachindependently H, halogen, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c); or

R⁷ taken together with R¹⁴ are part of an optionally substituted 5- or6-membered ring;

R⁸ taken together with R¹⁵ are part of an optionally substituted 5- or6-membered ring;

R⁹ taken together with R¹¹ are part of an optionally substituted 5- or6-membered ring;

R¹⁰ taken together with R¹⁶ are part of an optionally substituted 5- or6-membered ring;

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) an organic solvent; and

c) a desiccant.

In certain embodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). Incertain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are each independently H,alkyl, substituted alkyl, or sulfoalkyl. In certain embodiments, R⁷, R⁸,R⁹, and R¹⁰ are each independently a C₁-C₆ alkyl, which can be the sameor different. In certain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are eachmethyl. In certain embodiments, R¹² and R¹³ are each alkyl, preferably aC₁-C₆ alkyl. In certain embodiments, R¹² and R¹³ are each methyl. Incertain embodiments, R¹¹ and R¹⁴ are each independently alkyl,substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certainembodiments, R¹¹ and R¹⁴ are each independently a C₁-C₆ alkyl, a C₁-C₆substituted alkyl, or sulfoalkyl, which can be the same or different. Incertain embodiments, R_(x) is succinimidyl ester (SE),sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP) ester,tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a kit for tracking cell proliferation,differentiation and/or function, the kit being compatible for use with,for example, flow cytometry and fluorescence microscopy, and comprising:

a) a compound of structural formula (II):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl,-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹ and R²²are each independently H, halogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) an organic solvent; and

c) a desiccant.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R³, R⁴, R⁵ and R⁶ are each independently H or halo. Incertain embodiments, R³, R⁴, R⁵ and R⁶ are independently H, Cl, or F. Incertain embodiments, R⁷ and R⁹ are each independently alkyl, substitutedalkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c). In certain embodiments,R⁷ and R⁹ are each independently methyl, -L-R_(x), -L-S_(c) or(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 3. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a kit for tracking cell proliferation,differentiation and/or function, the kit being compatible for use with,for example, flow cytometry and fluorescence microscopy, and comprising:

a) a compound of structural formula (III):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl,-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³,and R²⁴ are each independently H, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) an organic solvent; and

c) a desiccant.

In certain embodiments, R¹² and R¹³ are each alkyl, preferably a C₁-C₆alkyl. In certain embodiments, R¹² and R¹³ are each methyl. In certainembodiments, R² is carboxyl, -L-R_(x), or -L-S_(c). In certainembodiments, R⁷, R⁹, R⁷, R¹⁹, R²⁰, R²², R²³ and R²⁴ are eachindependently alkyl, substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or-L-S_(c). In certain embodiments, R⁷, R⁹, R¹⁷, R²², R²³ and R²⁴ are eachmethyl. In certain embodiments, R¹⁹ and R²⁰ are each independently(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6. In certainembodiments, n is 1. In certain embodiments, R_(x) is succinimidyl ester(SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl (STP)ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP) ester,nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.

Another embodiment provides a kit for tracking cell proliferation,differentiation and/or function, the kit being compatible for use with,for example, flow cytometry and fluorescence microscopy, and comprising:

a) a compound of structural formula (IV):

or a pharmaceutically acceptable salt thereof, wherein

R¹² and R¹³ are each independently H, alkyl, substituted alkyl,-L-R_(x), or -L-S_(c);

R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹¹, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³,and R²⁴ are each independently H, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substitutedamino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c);

L is a linker;

R_(x) is a reactive group; and

S_(c) is a conjugated substance;

b) an organic solvent; and

c) a desiccant.

In certain embodiments, R⁹, R¹², R¹³, R²² and R²³ are each alkyl,preferably a C₁-C₆ alkyl.

In certain embodiments, R⁹, R¹², R¹³, R²² and R²³ are each methyl. Incertain embodiments, R³, R⁴, R⁵ and R⁶ are each independently H or halo.In certain embodiments, R³ and R⁶ are independently H, Cl, or F. Incertain embodiments, R² is carboxyl. In certain embodiments, R⁷, R⁹ andR² are each independently alkyl, substituted alkyl, sulfoalkyl, sulfo,(CH₂)_(n)SO₃ ⁻, wherein n is an integer between 1 and 6, -L-R_(x), or-L-S_(c). In certain embodiments, R⁷ is -L-R_(x) and R²⁰ is (CH₂)_(n)SO₃⁻. In certain embodiments, n is 1. In certain embodiments, R_(x) issuccinimidyl ester (SE), sulfodichlorophenyl (SDP) ester,sulfotetrafluorophenyl (STP) ester, tetrafluorophenyl (TFP) ester,pentaflurophenyl (PFP) ester, nitrilotriacetic acid (NTA), aminodextran,and cyclooctyne-amine.

In certain embodiments, R_(x) is selected from an acrylamide, anactivated ester of a carboxylic acid, a carboxylic ester, an acyl azide,an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline,an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a photoactivatable group, a reactive platinum complex,a silyl halide, a sulfonyl halide, and a thiol. In certain embodimentsthe reactive group is selected from the group consisting of carboxylicacid, succinimidyl ester of a carboxylic acid, hydrazide, amine and amaleimide. The reactive group may be attached to any appropriate site onthe reporter molecule or the aniline moiety. In certain embodiments, atleast one member selected from R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹²,R¹³, R¹⁹ and R²⁰ is a reactive group. In certain embodiments, at leastone of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is a reactive group. Incertain embodiments, at least one of R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ is areactive group.

In certain embodiments, S_(c) is selected from a carrier molecule and asolid support. In certain embodiments, S_(c) is selected from an aminoacid, a polymer of amino acids, a peptide, a protein, a neurotoxin, aphallotoxin, a cytokine, a toxin, a protease substrate, a protein kinasesubstrate, an enzyme, an antibody, an antibody fragment, a lectin, aglycoprotein, a histone, an albumin, a lipoprotein, avidin,streptavidin, protein A, protein G, a phycobiliprotein, a fluorescentprotein, a hormone, a growth factor, a nucleic acid base, a nucleoside,a nucleotide, a nucleic acid polymer, a nucleotide analog, a nucleosideanalog, a nucleoside triphosphate, a deoxynucleoside triphosphate, adideoxynucleoside triphosphate, a hapten, a carbohydrate, apolysaccharide, a lipid, an ion-complexing moiety (such as a crownether), a PEG group, and an organic or inorganic polymer. In certainembodiments, at least one member selected from R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁹ and R²⁰ is a conjugated substance. In certainembodiments, at least one of R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹² and R¹³ isa conjugated substance. In certain embodiments, at least one of R⁷, R⁸,R⁹, R¹⁰, R¹² and R¹³ is a conjugated substance. In certain embodiments,at least one of R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴,R¹⁵ and R¹⁶ are a PEG group.

In certain embodiments, the compounds used in the kits provided hereinare selected from the group consisting of Compound 1, Compound 2,Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8,Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19,Compound 20, Compound 21, Compound 22, Compound 23, Compound 24,Compound 25, and amine-reactive forms thereof.

In another illustrative embodiment of the kit, R_(X) is a succinimidylester. In another illustrative embodiment, the organic solvent is DMSO.

In certain embodiments, kits for tracking cell proliferation,differentiation and/or function are provided, the kit comprising:

(a) one or more of the compounds described herein;

(b) one or more containers; and optionally

(c) instructions for tracking cell proliferation, differentiation,and/or function according to a method disclosed herein.

In certain embodiments, kits are provided, the kit comprising:

(a) one or more of the compounds described herein;

(b) one or more containers; and optionally

(c) instructions for using according to a method disclosed herein.

In certain embodiments, the kits further comprise one or more of thefollowing: a buffering agent, a purification medium, a vial comprisingthe sample, or an organic solvent.

As used herein, the term “kit” refers to a packaged set of relatedcomponents, typically one or more dye compounds or compositions. Incertain embodiments, the kits disclosed herein comprise one or more ofthe dye compounds described herein, one or more carriers suitable for invitro or in vivo applications, and one or more containers in which tostore the one or more dye compounds and/or one or more carriers, such assolvents, buffers, stabilizers, pH adjusting agents, etc. The kitoptionally contains instructions for how to prepare the one or more dyecompounds or how to prepare a composition containing the one or more dyecompounds, and how to administer the dye compound or compositioncontaining the dye compound. In certain embodiments, the kit comprisesinstructions for performing an assay that tracks cellular proliferation,differentiation, and/or function. In certain embodiments, the assay isan in vitro assay. In certain embodiments, the assay is an in vivoassay. The kit may further comprise one or more pieces of equipment toadminister the compound, or composition containing the compoundincluding, but not limited to, syringes, pipettes, pipette bulbs,spatulas, vials, syringe needles, and various combinations thereof.

In certain embodiments, the kits provided herein comprise indicatorsolutions or indicator “dipsticks”, blotters, culture media, cuvettes,and the like. In certain embodiments, the kits provided herein compriseindicator cartridges (where a kit component is bound to a solid support)for use in an automated detector. In certain embodiments, the kitsprovided herein further comprise molecular weight markers, wherein saidmarkers are selected from phosphorylated and non-phosphorylatedpolypeptides, calcium-binding and non-calcium binding polypeptides,sulfonated and non-sulfonated polypeptides, and sialylated andnon-sialylated polypeptides. In certain embodiments, the kits providedherein further comprise a member selected from a fixing solution, adetection reagent, a standard, a wash solution, and combinationsthereof.

In certain embodiments, kits for tracking cell proliferation,differentiation and/or function are provided, the kit comprising:

(a) one or more of the compositions described herein;

(b) one or more containers; and optionally

(c) instructions for tracking cell proliferation, differentiation,and/or function according to a method disclosed herein.

A detailed description of the present teachings having been providedabove, the following examples are given for the purpose of illustratingthe present teachings and shall not be construed as being a limitationon the scope of the disclosure or claims.

EXAMPLES Example 1: Chemical Synthesis of Cell Tracking Compounds A.Synthesis of Compound 2:

1,2,3,4-tetrahydroquinoline (10 g, 75.2 mmol)) was dissolved in ethanol(100 mL). 20 mL of 37% formaldehyde was added, followed by addition ofNaCNBH₃ (9.4 g) and 1 mL of AcOH. The reaction was stirred for 18 hours.The reaction mixture was treated with 100 mL of saturated ammoniumchloride and stirred for 1 hour. After removal of ethanol in vacuo, theremaining solution was extracted with 200 mL of dichloromethane twice.The combined organic layer was washed with saturated NaCl, dried oversodium sulfate and concentrated in vacuo. The residue was purified byflash chromatograph with hexane-ethyl acetate to afford1-methyl-1,2,3,4-tetrahydroquinoline as a clear oil (yield: 90%). ¹H NMR(CDCl₃): δ 7.13 (t, 1H), 7.0 (t, 1H), 6.63 (m, 2H), 3.26 (t, 2H), 2.90(s, 3H), 2.80 (m, 2H), 2.01 (t, 2H).

Phthalic anhydride (9.6 g, 65.3 mmol), anhydrous aluminum chloride (10.8g, 80.9 mmol), and dry dichloromethane (100 mL) were added to a 250 mLof flask. The mixture was stirred for 1 hour at room temperature andcooled in an ice bath. The solution of1-methyl-1,2,3,4-tetrahydroquinoline (8.0 g, 60.1 mmol) dissolved in 40mL of dichloromethane was added dropwise. After the addition wascomplete, the reaction was stirred for 4 hours at room temperature. Thereaction mixture was poured into ice-cold HCl solution, and extractedwith dichloromethane. The organic layer was collected and dried oversodium sulfate. It was purified by column chromatography withdichloromethane-methanol on silica gel to yield2-(1-methyl-1,2,3,4-tetrahydroquinoline-6-carbonyl)benzoic acid (yield:30%). ¹H NMR (d₆-DMSO): δ 7.90 (s, 1H), 7.67 (m, 2H), 7.32 (m, 2H), 6.50(m, 2H), 3.20 (t, 2H), 2.98 (s, 3H), 2.65 (m, 2H), 1.89 (t, 2H)

7-bromo-1,2,3,4-tetrahydroquinoline (5.0 g, 23.6 mmol) was dissolved inDMF (10 mL). Potassium carbonate (6.4 g) and 6-bromohexanoate ethylester (10.0 g, 47.2 mmol) were added into the solution. The mixture washeated to 100° C. for 18 hours. After the reaction was cooled down, 50mL of dichloromethane was added and washed with 10 mL of water threetimes. The organic layer was dried over sodium sulfate and concentrated.It was purified by flash chromatography in silica gel with hexanes-ethylacetate to afford ethyl6-(7-bromo-3,4-dihydroquinolin-1(2H)-yl)hexanoates as a clear oil(yield: 70%). ¹H NMR (CDCl₃): δ 6.79 (d, 1H), 6.64 (d, 2H), 4.15 (t,2H), 3.29 (t, 2H), 3.21 (t, 2H), 2.32 (t, 2H), 1.92 (m, 2H), 1.72 (m,2H), 1.65 (m, 2H), 1.43 (m, 2H), 1.31 (t, 3H).

Ethyl 6-(7-bromo-3,4-dihydroquinolin-1(2H)-yl)hexanoate (4.0 g, 11.3mmol), isopropenylboronic acid pinacol ester (2.7 g, 16.1 mmol),palladium acetate (253 mg), and potassium carbonate (4.65 g) were mixedwith 50 mL of toluene, 50 mL of isopropanol, and 50 mL of water. Themixture was bubbled with argon for 10 min. The mixture was heated to100° C. for 16 hours.

After removal of organic solvents in vacuo, the residue was extractedwith dichloromethane three times. The combined organic layer was driedover sodium sulfate and concentrated. It was purified by flashchromatography with hexane-ethyl acetate to afford ethyl6-(7-(prop-1-en-2-yl)-3,4-dihydroquinolin-1(2H)-yl)hexanoate as lightyellow oil (yield: 67%). ¹H NMR (CDCl₃): δ 6.92 (d, 1H), 6.68 (m, 2H),5.28 (s, 1H), 5.01 (s, 1H), 4.14 (t, 2H), 3.30 (m, 4H), 2.74 (t, 2H),2.32 (t, 2H), 2.12 (s, 3H), 1.95 (m, 2H), 1.66 (m, 4H), 1.42 (m, 2H),1.29 (t, 3H).

2-(1-methyl-1,2,3,4-tetrahydroquinoline-6-carbonyl)benzoic acid (3.9 g,13.2 mmol), and ethyl6-(7-(prop-1-en-2-yl)-3,4-dihydroquinolin-1(2H)-yl)hexanoate (4.0 g,12.7 mmol) were dissolved in dry dichloromethane (100 mL). P₂O₅ (9.0 g)was added into the stirred solution. The mixture was refluxed for 1hour. After removal of solvent, the residue was treated with sulfuricacid (10 mL). The reaction was stirred for 18 hours. It was poured intoice and extracted with dichloromethane three times. The combined organiclayer was dried over sodium sulfate and concentrated. It was purified byflash chromatography with 15% water in acetonitrile to afford2-(1-(5-carboxypentyl)-11-metheyliumyl-13,13-dimethyl-1,2,3,4,8,9,10,13-octahydro-1114-benzo[1,2-g:5,4-g′]diquinolin-6-yl)benzoate(Compound 1) as a dark blue solid (yield: 9%). ¹H NMR (MeOD): δ 8.29 (d,1H), 7.81 (t, 1H), 7.74 (t, 1H), 7.29 (d, 1H), 7.10 (d, 1H), 6.65 (d,1H), 3.71 (t, 2H), 3.60 (m, 4H), 2.55 (m, 4H), 2.41 (t, 2H), 1.92 (m,4H), 1.70 (m, 10H), 1.52 (m, 2H).

2-(1-(5-carboxypentyl)-11-metheyliumyl-13,13-dimethyl-1,2,3,4,8,9,10,13-octahydro-1114-benzo[1,2-g:5,4-g′]diquinolin-6-yl)benzoate(600 mg, 1.06 mmol) was dissolved in dry DMF (10 mL). DIEA (0.36 mL) andTSTU (340 mg) were added to the solution. The reaction was stirred for 1hour and was added dropwise to ethyl ether (100 mL). The mixture wascentrifuged to afford2-(1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-11-metheyliumyl-13,13-dimethyl-1,2,3,4,8,9,10,13-octahydro-1114-benzo[1,2-g:5,4-g′]diquinolin-6-yl)benzoate(Compound 2) after filtration from ethyl ether as a dark blue powder(yield: 92%). ¹H NMR (MeOD): δ 8.26 (d, 1H), 7.80 (t, 1H), 7.73 (t, 1H),7.30 (d, 1H), 7.12 (d, 2H), 6.63 (d, 2H), 3.70 (t, 2H), 3.62 (m, 4H),2.80 (t, 4H), 2.70 (m, 4H), 2.60 (t, 2H), 1.94 (m, 4H), 1.72 (m, 10H),1.50 (m, 2H).

B. Synthesis of Compound 3:

3,6-dichlorophthalic anhydride (0.5 g, 2.30 mmol), anhydrous aluminumchloride (0.6 g, 4.5 mmol), dry dichloromethane (10 mL) were added to a100 mL flask. The mixture was stirred for 1 hour at room temperature andcooled in an ice bath. A solution of1-methyl-1,2,3,4-tetrahydroquinoline (0.40 g) dissolved in 10 mL ofdichloromethane was added dropwise. After the addition was complete, thereaction was stirred for 4 hours at room temperature. The reactionmixture was poured into an ice cold HCl solution, and extracted withdichloromethane. The organic layer was collected and dried over sodiumsulfate. It was purified by column chromatography with 5% methanol indichloromethane on silica gel to yield3,6-dichloro-2-(1-methyl-1,2,3,4-tetrahydroquinoline-6-carbonyl)benzoicacid (yield: 30%). ¹H NMR (d₆-DMSO): δ 7.62 (s, 1H), 6.91 (m, 1H), 6.82(m, 1H), 6.50 (m, 2H), 3.14 (t, 2H), 2.91 (s, 3H), 2.69 (m, 2H), 1.85(t, 2H).

3,6-dichloro-2-(1-methyl-1,2,3,4-tetrahydroquinoline-6-carbonyl)benzoicacid (0.10 g, 0.27 mmol) and ethyl6-(7-(prop-1-en-2-yl)-3,4-dihydroquinolin-1(2H)-yl)hexanoate (0.088 g,0.27 mmol) were dissolved in dry dichloromethane (5 mL). P₂O₅ (0.200 g)was added into the stirred solution. The mixture was refluxed for 1hour. After removal of solvent, the residue was treated with sulfuricacid (3 mL). The reaction was stirred for 18 hours. It was poured intoice and extracted with dichloromethane three times. The combined organiclayer was dried over sodium sulfate and concentrated. It was purified byflash chromatography with 5% methanol in dichloromethane on silica gelto afford2-(1-(5-carboxypentyl)-11-metheyliumyl-13,13-dimethyl-1,2,3,4,8,9,10,13-octahydro-1114-benzo[1,2-g:5,4-g′]diquinolin-6-yl)-3,6-dichlorobenzoateas a dark blue solid (yield: 9%). LCMS: 634.6 (M+1)

2-(1-(5-carboxypentyl)-11,13,13-trimethyl-2,3,4,8,9,10,11,13-octahydrobenzo[1,2-g:5,4-g′]diquinolin-1-ium-6-yl)-3,6-dichlorobenzoate(20 mg, 0.032 mmol) was dissolved in dry DMF (1 mL). TEA (0.10 mL) andTSTU (15 mg) were added to the solution. The reaction was stirred for 1hour and added dropwise to ethyl ether (100 mL). The mixture wascentrifuged to afford3,6-dichloro-2-(1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-11-metheyliumyl-13,13-dimethyl-1,2,3,4,8,9,10,13-octahydro-1114-benzo[1,2-g:5,4-g′]diquinolin-6-yl)benzoate(Compound 3) after filtration of ethyl ether as dark blue powder (yield:90%). ¹H NMR (MeOD): δ 8.20 (d, 1H), 7.28 (d, 1H), 7.11 (d, 2H), 6.58(d, 2H), 3.69 (t, 2H), 3.62 (m, 4H), 2.80 (t, 4H), 2.69 (m, 4H), 2.58(t, 2H), 1.94 (m, 4H), 1.72 (m, 10H), 1.50 (m, 2H), LCMS: 731.7 (M+1).

C. Synthesis of Compound 18:

Aniline (2 g, 21.5 mmol) was dissolved in THF (20 mL) and cooled to 0°C. in an ice bath. TFA (1 eq) was added into the solution and stirredfor 15 min. Mesityl oxide (4-methyl-3-penten-2-one) (1.2 eq) was addedinto the mixture, which was allowed to warm up to room temperature. Thereaction was stirred overnight. After THF was removed in vacuo, theresidue was suspended in CHCl₃ (˜2 L), and mechanically stirred until auniform solution was achieved. H₂O (˜1.5 L) was added to the solution,and the pH was adjusted to 8-9 using solid KOH. The organic phase waswashed with H₂O (3×˜1 L) and brine (˜1 L), and dried over anhydroussodium sulfate (Na₂SO₄). After CHCl₃ was removed in vacuo, the residuewas dissolved in ethanol (20 mL), formaldehyde (37%, 2 mL) was added,followed by addition of NaCNBH₃ (3.5 g) and 1 mL of AcOH. The reactionwas stirred for 18 hours. The reaction mixture was treated with 100 mLof saturated ammonium chloride and stirred for 1 hour. After removal ofethanol in vacuo, the remaining solution was extracted with 200 mL ofdichloromethane twice. The combined organic layer was washed withsaturated NaCl, dried over sodium sulfate and concentrated in vacuo. Theresidue was purified by flash chromatograph with hexane-ethyl acetate toafford 1,2,2,4-tetramethyl-1,2-dihydroquinoline as a light yellow oil(yield: 70%). ¹H NMR (CDCl₃): δ 7.23 (t, 2H), 6.65 (m, 2H), 5.26 (s,1H), 2.80 (s, 3H), 2.00 (s, 3H), 1.32 (s, 3H).

3,6-dichlorophthalic anhydride (5.0 g, 23 mmol), anhydrous aluminumchloride (5.8 g, 43.6 mmol), and dry dichloromethane (100 mL) were addedto a 250 mL flask. The mixture was stirred for 1 hour at roomtemperature and cooled in an ice bath. A solution of1,2,2,4-tetramethyl-1,2-dihydroquinoline (4.50 g, 24 mmol) dissolved in40 mL of dichloromethane was added dropwise. After the addition wascomplete, the reaction was stirred for 4 hours at room temperature. Thereaction mixture was poured into ice cold HCl solution, and extractedwith dichloromethane. The organic layer was collected and dried oversodium sulfate. It was purified by column chromatography withdichloromethane-methanol on silica gel to yield3,6-dichloro-2-(1,2,2,4-tetramethyl-1,2-dihydroquinoline-6-carbonyl)benzoicacid (yield: 30%). LCMS: 404.29.

3,6-dichloro-2-(1,2,2,4-tetramethyl-1,2-dihydroquinoline-6-carbonyl)benzoicacid (1.0 g, 2.47 mmol) and ethyl6-(7-(prop-1-en-2-yl)-3,4-dihydroquinolin-1(2H)-yl)hexanoate (0.8 g,2.53 mmol) were dissolved in dry dichloromethane (30 mL). P₂O₅ (3.0 g)was added into the stirred solution. The mixture was refluxed for 1hour. After removal of solvent, the residue was treated with sulfuricacid (10 mL). The reaction was stirred for 18 hours. It was poured intoice and extracted with dichloromethane three times. The combined organiclayer was dried over sodium sulfate and concentrated. It was purified byflash chromatography with 15% water in acetonitrile on silica gel toafford2-(1-(5-carboxypentyl)-11-metheyliumyl-2,2,13,13-tetramethyl-4-(sulfomethyl)-1,2,8,9,10,13-hexahydro-1114-benzo[1,2-g:5,4-g′]diquinolin-6-yl)-3,6-dichlorobenzoateas a dark blue solid (yield: 10%). LCMS: 752.72.

2-(11-(5-carboxypentan-1-ylium-1-yl)-1,2,2,13,13-pentamethyl-4-(sulfomethyl)-1,2,8,9,10,13-hexahydro-11λ4-benzo[1,2-g:5,4-g′]diquinolin-6-yl)-3,6-dichlorobenzoate(50 mg, 0.066 mmol) was dissolved in dry DMF (3 mL). DIEA (0.04 mL) andTSTU (60 mg) were added to the solution. The reaction was stirred for 1hour and added dropwise to ethyl ether (50 mL). The mixture wascentrifuged to afford3,6-dichloro-2-(1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-11-metheyliumyl-2,2,13,13-tetramethyl-4-(sulfomethyl)-1,2,8,9,10,13-hexahydro-1114-benzo[1,2-g:5,4-g′]diquinolin-6-yl)benzoate(Compound 18) after filtration from ethyl ether as a dark blue powder(yield: 90%). LCMS: 849.80.

Example 2: Cell Tracking Activity of Compounds 2.3 and 12

The whole blood sample (20 mL) was diluted in 20 mL of PBS and thencentrifuged for 30 min after addition of FICOLL-PAQUE Plus (15 mL) (GEHealthcare). The lymphocyte layer of cells was suspended in 25 mL of PBSbuffer and centrifuged for 5 min. The cells were counted on the COUNTESSAutomated Counter and the concentration was adjusted to 10⁶ cells/mL. 10mL of cells was incubated with either Compound 2 (FIG. 1A) or CELLTRACEFar Red DDAO (FIG. 1B) (Thermo Fisher Scientific) (5 μM) for 20 min in a37° C. water bath. After incubation the cells were treated with 40 mL ofOPTMIZER T Cell Expansion (Thermo Fisher Scientific) for 5 min. Thetreatment with OPTMIZER T Cell Expansion was repeated one more timeafter centrifugation. Next, the stained cells were stimulated with 50 μLDYNABEADS Human T-Activator CD3/CD28 (Thermo Fisher Scientific) per 1 mLof cells for 5 days. The stimulated cells were then analyzed using anATTUNE flow cytometer (Thermo Fisher Scientific) with 633/635 nmexcitation and a 660/20 bandpass emission filter.

Cell tracing by dye dilution was assessed by staining PBMCs isolated byFICOLL-PAQUE (GE Healthcare) density gradient centrifugation. Aliquotsof isolated PBMCs were stained with Compound 3 or Compound 12, splitinto samples for culturing and one sample was stimulated with anti hCD3(clone UCHT1) and supplemented with recombinant hIL2. Dye loading wasobserved by fluorescent microscopy. After proliferation for 3 to 5 days,samples were stained with an appropriate viability dye (e.g., a SYTOXdye (Thermo Fisher Scientific)) and analyzed by flow cytometry.Permeability was assessed by imaging. Toxicity was assessed byinterrogation with the SYTOX dye. The results showing passage of thedyes through multiple cell generations are shown in FIGS. 2A and 2B(Compound 12) and FIGS. 3A and 3B (Compound 3). FIGS. 2A and 3A show adual parameter plot showing the live cells (within the box) and FIGS. 2Band 3B show the generational peaks (6-8 generations) as determined byflow cytometry.

Briefly, for the separation of mononuclear cells from whole blood: 20 mLof whole blood were added to 20 mL DPBS and mixed well. 15 mLFICOLL-PAQUE PLUS (GE Healthcare) were added to each of two 50 mLcentrifuge tubes. 20 mL of the diluted whole blood was carefully layeredon top and centrifuged for 30 min at 400× g with the brake off. The PBMClayer was carefully removed. Cells were resuspended in 25 mL DPBS in a50 mL centrifuge tube and were centrifuged for 5 min at 400× g. Thesupernatant was decanted and the cells were resuspended in 10 mL DPBS.Cells were counted a COUNTESS Automated Cell Counter (Thermo FisherScientific) (can also be counted on another counting device) and theconcentration was adjusted to 1×10⁶ cells/mL in DPBS.

To stain the cells, the cells were labeled with the appropriate dyecompound (Compound 3 or 12, stock solution was made by addingapproximately 1 L per mL of cell suspension). The tubes were immediatelyvortexed for 30 sec and the cells were incubated for 20 min at roomtemperature, protected from light. Occasionally swirling the cells toprevent settling helped to produce more uniform labeling. 5 times thevolume of stained cells of prepared OPTMIZER CTS T-Cell Expansion SFMmedium (Thermo Fisher Scientific) was added and incubated 5 min at roomtemperature, protected from light. The cells were pelleted bycentrifuging 5 min at 400× g, then pouring off the supernatant. The cellpellet was resuspended in 2 mL DPBS+10% FBS. The cells were pelletedagain by centrifuging 5 min at 400× g, then pouring off the supernatant.The cell pellet was resuspended in 10 mL of prepared OPTMIZER CTS T-CellExpansion SFM medium.

Aliquots of the stained cells were distributed into a 6-well or 24-wellculture plate (at least 1 mL per well).

For stimulating and culturing T lymphocytes for proliferation analysis,the cells were stimulated with one of the following treatments:

-   -   1. 1 μg anti-hCD3 (2 μL of UCHT) and 100 ng IL-2 (1 μL of 0.1        mg/mL solution); or    -   2. 50 μL CD3/CD28 DYNABEADS (Thermo Fisher Scientific) Human        T-Activator CD3/CD28 T cell expander beads.

For determination of cell permeability, PBMCs were isolated byFicoll-Pacque density gradient centrifugation and stained with theappropriate dye compound (e.g., Compound 2). The samples were split intosamples for culturing and one sample was stimulated with anti-hCD3(clone UCHT1) and supplemented with recombinant hIL2. Dye loading wasobserved by fluorescent microscopy. After proliferation for 3 to 5 days,the samples were stained with the appropriate viability dye (e.g., SYTOXdye) and analyzed by flow cytometry (see FIG. 3).

To test for cytotoxicity, PBMCs were stained with CELLTRACE (ThermoFisher Scientific) CFSE (positive control) or Compound 2. The cells weresplit into samples for culturing and one sample was stimulated withanti-hCD3 (clone UCHT1) and supplemented with recombinant hIL2. Dyeloading was observed by fluorescent microscopy. After proliferation for3 to 5 days, the samples were stained with the appropriate viability dye(e.g., SYTOX dye) and analyzed by flow cytometry (see Table 3).

TABLE 3 Cytotoxicity Analysis of Compound 2 Dye Compound % Live CellsCELLTRACE CFSE 52 Compound 2, 2 μM 53 Compound 2, 5 μM 55 Compound 2, 10μM 64

Example 3: Wound Healing Activity Analysis

Neonatal human dermal fibroblasts were grown to confluency and thenlabeled with 1 μM Compound 2 for 30 minutes in LCIS. LCIS removed andmedia was placed back on cells. Scratch wound was made in the cells. Theentire wound was scanned on EVOS Auto FL (Thermo Fisher Scientific)using the scan function. Same area was re-scanned 24 and 48 hours laterto monitor how the wound was healing. The initial scan of the freshwound was analyzed using the measure function on the EVOS Auto FL tocalculate the area of the wound (7.25×10⁶ μm²). Results are shown inFIG. 5.

Example 4: Angiogenesis Analysis

Angiogenesis: Human umbilical vein endothelial cells were plated onGELTREX (Thermo Fisher Scientific) coated plates at a density of 35,000cells/cm² and incubated for 45 minutes. The cells were then labeled with1 μM Compound 2 for 30 minutes in LCIS. LCIS removed and M-200 mediawith LVES was placed back on cells. Cells were incubated at 37° C. with5% CO₂ for 20 hours and imaged. The results are shown in FIG. 6.

Example 5: Cell Differentiation Analysis

Human Skeletal Myoblast Differentiation: Human skeletal myoblast wereplated on 24 well plates at a density of 240,000 cells/well. Cells wereincubated for 4 hours in DMEM+20% FBS to let the cells attach to theplate. Next, cells were labeled with 1 μM Compound 2 for 30 minutes inLCIS. LCIS removed and low glucose DMEM media with 2% horse serum wasplaced on cells. Cells were incubated for 44 hours to let the cellsdifferentiate into muscle fibers. Cells were then imaged. The resultsare shown in FIG. 7.

Example 6: Reaction of Goat Anti-Mouse IgG (GAM) and SulfonatedCarbopyronine (Compound 18

0.12 mL (1 mg) of an 8.4 mg/mL solution of GAM in 10 mM potassiumphosphate, 150 mM sodium chloride buffer (PBS) was measured into aplastic vial and the pH raised to 8.5-9.0 with 12 μL 1 M sodiumbicarbonate, pH 9.0. The GAM solution was reacted with a 5-fold molarexcess of the sulfonated carbopyronine (Compound 18) at 10 mg/mL inanhydrous DMSO for 1 h at room temperature while stirring gently. Thedye-protein conjugate was separated from free dye by size exclusionchromatography using a 0.75×20 cm column packed with BIO-RAD BIO-GELP-30 (Bio-Rad Laboratories, Inc.) fine in PBS and eluted with same. Theinitial protein-containing band from the column was collected. Theabsorbance spectrum was measured using a PerkinElmer Lambda 35 UV/Visspectrometer. The fluorescence emission spectrum was obtained using aPerkinElmer LS 55 Fluorescence Spectrometer, excited at 610 nm.

As shown in FIG. 8, the DOS for the conjugated form of Compound 18 is3.7, whereas the DOS for the conjugated form of a similarly colored dye(ALEXA FLUOR 680) is 3.9. At these similar DOS levels, the Compound18-GAM conjugate brightness is >2× that of the ALEXA FLUOR 680-GAMconjugate.

Example 7: Bead Staining with Compound 25

To 0.7 mL of a 5.2 μM PEGylated latex beads solution, add 0.35 mLmethanol and stir at room temperature for five minutes, followed byadding 0.8 mg Compound 25 dissolved in 540 μL dichloromethane and 125 μLethanol solution drop by drop. Continue to stir the bead stainingsolution at room temperature for 30 minutes followed by removal oforganic solvent from the bead solution with a rotovap. Finally, filterthe bead solution through a 0.2 μm filter to obtain the stained latexbeads.

The stained beads have an emission maximum at 690 nm, 15% quantum yieldwhen excited at 633 nm, and about 4.5×10⁶ M⁻¹ cm⁻¹ extinctioncoefficient at 633 nm.

Example 8: Reaction of Allophycocyanin (APC) and Compound 18,Succinimidyl Ester (SE)

0.3 mL (3 mg) of a 10 mg/mL solution of APC in 0.1 M sodium phosphate,0.1 M sodium chloride buffer, pH 7.5 was reacted with a 5, 10, or120-fold molar excess of Compound 18 at 10 mg/mL in anhydrousdimethylsulfoxide (DMSO) for 1.5 hours at room temperature (RT). Thedye-protein conjugates were separated from free dye by size exclusionchromatography using 3-1.5×10 cm columns packed with BIORAD BIO-GEL P-30fine in 0.1M sodium phosphate, 0.1 M sodium chloride buffer, pH 7.5 andeluted with same.

The initial protein-containing band from each column was collected. Theabsorbance spectra were obtained on a PerkinElmer Lambda 35 UV/VISspectrometer. The fluorescence emission spectra were obtained using aPerkinElmer LS55 Fluorescence Spectrometer, excited at 633 nm. Theconjugates show a shift in emission from 660 nm to 690 nm compared tounmodified APC excited at the same wavelength.

The complex may be further modified by the conjugation of approximately1-2 moles of SPDP (3-(2-pyridyldithio) propionic acid succinimidylester), to give a pyridyldisulfide-modified protein. Following reductionof the disulfide, such as with dithiothreitol (DT), this thiolatedprotein is readily conjugated to any thiol-reactive second protein, suchas mouse anti-human CD4 that has been modified by SMCC(4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid succinimidylester). The labeled mouse anti-human CD4 is then typically purifiedusing size-exclusion chromatography. Covalent conjugates of moleculesother than proteins, such as oligonucleotides, nucleic acids,microspheres, liposomes and so forth are readily prepared by similarmethods using the same or other crosslinking reagents known in the art.

Flow Cytometry:

Candidates were conjugated to mouse monoclonal antibody against humanCD4 (clone S3.5). White blood cells were prepared from ammoniumchloride-lysed whole blood. Antibody conjugates, made with experimentalcompounds or with reference fluorochromes, were diluted in 1% BSA inPBS, pH7.4, and 10 uL of the conjugates were added to 90 μL of 1×10⁶cells. Conjugates and cells were allowed to incubate for 20 minutes atroom temperature. After which, the cells were washed 2 times in 1% BSAin PBS, pH7.4. After the final wash, the cells were resuspended in 500uL of 1% BSA in PBS, pH7.4. Stained cells were analyzed using the ATTUNEAcoustic Focusing Cytometer (Thermo Fisher Scientific). For CD4 specificstaining, 10,000 lymphocyte events were collected, and data wasrepresented as a histogram. Bi-Markers or histogram markers wereinserted to help determine the percentage of CD4⁺ cells in thelymphocyte population. This was used as criteria to determine if therewere any effects of the fluorochrome, conjugation methodology on theantibody performance. In addition, values from the mean fluorescenceintensities of both the negative and positive peaks, as well as thestandard deviation of the negative peak were used to determine thestaining index of the conjugate tested. Comparison of the stainingindexes was used to aid in determining the performance of differentconjugates.

As shown in FIG. 9A, the flow cytometry histograms showing signal fromlymphocytes stained with APC-CD4 conjugate in four emission channels:BL1-H (530/30 nm), BL2-H (574/26 nm), BL3 (690/50 nm), BL4 (780/60), RL1(660/20 nm), RL2-H (780/60 nm). FIG. 9B shows flow cytometry histogramsshowing signal from lymphocytes stained with Compound 18-CD4 conjugatein four emission channels: BL1-H (530/30 nm), BL2-H (574/26 nm), BL3(690/50 nm), BL4 (780/60 nm), RL1 (660/20 nm), RL2-H (780/60 nm). Notethe lack of spillover in BL3-H, BL4-H, and RL1-H, compared with thatfrom APC-CD4 in FIG. 9A; Compound 18-CD4 enables bright signalspecifically in RL2, in contrast to signal in multiple channels usingAPC-CD4. FIGS. 9C and 9D show flow cytometry histograms showing signalin the RL2-H channel (780/60 nm) from lymphocytes stained with tandemdye conjugates APC-ALEXA FLUOR 750-CD4 (left) and APC-Compound 18-CD4(right). FIG. 9E shows flow cytometry histograms showing signal fromlymphocytes stained with tandem dye conjugates of CD4 in the RL2-Hchannel (705/40 nm). Left histogram shows signal from cells stained withAPC-ALEXA FLUOR 750-CD4, middle histogram shows signal from APC-Compound18-CD4 DOL 5, and right histogram shows signal from APC-Compound 18-CD4DOL10. The term DOL refers to the number of molecules of Compound 18 pernumber of molecules of APC in the tandem conjugates. Table 4 shows theimproved SI (Staining Index) and Signal/Noise (S:N) of conjugates madewith Compound 18 over those made with ALEXA FLUOR 750 (Thermo FisherScientific).

TABLE 4 Staining Index and Signal: Noise Ratio of Compound 18 Conjugates705/40 nm Neg Pos SD SI S:N APC-AF750 968 41176 474 42 43 18-APC DOL 55632 1433019 3484 205 254 18-APC DOL 10 4973 700962 3149 111 141

Example 9: Cytotoxicity Analysis of Compound 2

FIG. 10 demonstrates the lack of cytotoxicity of Compound 2, as measuredby the CYQUANT Direct (Thermo Fisher Scientific) method. Fluorescence onthe Y-axis is proportional to cell population viability. A549 cellsplated in a 96 well plate were labeled with CELLTRACKER Blue, Green,Red, Compound 2, or DMSO in Live Cell Imaging Solution at 10 μMconcentration for 30 mins at 37° C. The LCIS was then aspirated fromcells and complete media added for an overnight incubation. Thefollowing day (Day 1) cells were assayed for cytotoxicity using theCYQUANT Direct assay (Thermo Fisher Scientific). The CYQUANT Directassay was repeated on cells from the same 96 well plate on days 2 and 3showing no cytotoxicity of CELLTRACKER Blue, Green, Red or Compound 2when compared to DMSO alone.

1. A compound of structural formula (I):

or a pharmaceutically acceptable salt thereof, wherein R¹² and R¹³ areeach independently H, alkyl, substituted alkyl, -L-R_(x), or -L-S_(c);R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵, and R¹⁶ are eachindependently H, halogen, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c); or R⁷ taken togetherwith R¹⁴ are part of an optionally substituted 5- or 6-membered ring; R⁸taken together with R¹⁵ are part of an optionally substituted 5- or6-membered ring; R⁹ taken together with R¹¹ are part of an optionallysubstituted 5- or 6-membered ring; R¹⁰ taken together with R¹⁶ are partof an optionally substituted 5- or 6-membered ring; L is a linker; R_(x)is a reactive group; and S_(c) is a conjugated substance. 2-6.(canceled)
 7. The compound according to claim 1, wherein R¹² and R¹³ areeach methyl.
 8. The compound according to claim 1, wherein R³, R⁴, R⁵and R⁶ are each independently H or halo.
 9. (canceled)
 10. The compoundaccording to claim 1, wherein R² is carboxyl, -L-R_(x), or -L-S_(c). 11.The compound of claim 1, wherein R¹¹ and R¹⁴ are each independentlyalkyl, substituted alkyl, sulfoalkyl, sulfo, -L-R_(x), or -L-S_(c).12-15. (canceled)
 16. The compound according to claim 1, wherein R_(X)is an acrylamide, a carboxylic acid, an activated ester of a carboxylicacid, an acyl azide, an acyl halide, hydroxy, an aldehyde, an alkylhalide, a sulfonate, an amine, an anhydride, an aniline, an aryl halide,an azide, an aziridine, a boronate, a carbodiimide, a diazoalkane, anepoxide, a glycol, a haloacetamide, a halomethyl, a halotriazine, ahydrazine, a hydroxylamine, an imido ester, an iodoacetamide, anisothiocyanate, a ketone, a maleimide, a sulfonyl halide, a thiol group,a succinimidyl ester (SE), a sulfodichlorophenyl (SDP) ester, asulfotetrafluorophenyl (STP) ester, a tetrafluorophenyl (TFP) ester, apentafluorophenyl (PFP) ester or a cyclooctyne-amine. 17-18. (canceled)19. The compound according to claim 1, wherein S_(c) is an amino acid, apeptide, a protein, a monosaccharide, a polysaccharide, anion-complexing moiety, a nucleotide, an oligonucleotide, a nucleic acid,a hapten, a drug, a toxin, a tyramide, a lipid, a phospholipid, alipoprotein, a lipopolysaccharide, a liposome, a lipophilic polymer, aPEG group, a non-biological organic polymer, a polymeric microparticle,an animal cell, a plant cell, a bacterium, a yeast, or a virus. 20-24.(canceled)
 25. A method for tracking cell proliferation,differentiation, and/or function, the method comprising the steps of: a)incubating a mixture of cells and a compound of structural formula (I):

or a pharmaceutically acceptable salt thereof, wherein R¹² and R¹³ areeach independently H, alkyl, substituted alkyl, -L-R_(x), or -L-S_(c);R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, R¹⁵, and R¹⁶ are eachindependently H, halogen, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester,(carboxyl ester) amino, (carboxyl ester) oxy, cyano, alkylcarboxylate,hydroxyl, nitro, sulfo, sulfoalkyl, substituted sulfoalkyl, sulfonyl,substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthiol,substituted alkylthiol, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, -L-R_(x), or -L-S_(c); or R⁷ taken togetherwith R¹⁴ are part of an optionally substituted 5- or 6-membered ring; R⁸taken together with R¹⁵ are part of an optionally substituted 5- or6-membered ring; R⁹ taken together with R¹¹ are part of an optionallysubstituted 5- or 6-membered ring; R¹⁰ taken together with R¹⁶ are partof an optionally substituted 5- or 6-membered ring; L is a linker; R_(x)is a reactive group; and S_(c) is a conjugated substance; b) providing astimulus to the mixture to elicit a fluorescent signal; and c) analyzingthe stimulated mixture. 26-28. (canceled)
 29. The method according toclaim 25, wherein the compound is selected from the group consisting of

and amine-reactive forms thereof.
 30. The method according to claim 25,further comprising a second compound excitable at a different wavelengththat the cell-tracking compound of structural formula (I).
 31. Themethod according to claim 30, wherein the second compound is CFDA-SE orGFP.
 32. The method according to claim 25, wherein R_(X) is anacrylamide, a carboxylic acid, an activated ester of a carboxylic acid,an acyl azide, an acyl halide, hydroxy, an aldehyde, an alkyl halide, asulfonate, an amine, an anhydride, an aniline, an aryl halide, an azide,an aziridine, a boronate, a carbodiimide, a diazoalkane, an epoxide, aglycol, a haloacetamide, a halomethyl, a halotriazine, a hydrazine, ahydroxylamine, an imido ester, an iodoacetamide, an isothiocyanate, aketone, a maleimide, a sulfonyl halide, or a thiol group.
 33. (canceled)34. The method according to claim 32, wherein R_(X) is a succinimidylester (SE), sulfodichlorophenyl (SDP) ester, sulfotetrafluorophenyl(STP) ester, tetrafluorophenyl (TFP) ester, pentafluorophenyl (PFP)ester, nitrilotriacetic acid (NTA), aminodextran, and cyclooctyne-amine.35. The method according to claim 25, wherein step a) is conducted forapproximately 20 minutes.
 36. The method according to claim 25, whereinstep b) and step c) are carried out concurrently.
 37. The methodaccording to claim 25, wherein step c) involves flow cytometry.
 38. Akit for tracking cell proliferation, differentiation, and/or function,the kit comprising: a) a compound of claim 1; b) an organic solvent; andc) a desiccant. 39-46. (canceled)
 47. A composition for tracking cellproliferation, differentiation, and/or function, the compositionscomprising: (a) one or more of the compounds of claim 1; and b) acarrier, wherein the one or more of the compounds are present in anamount effective to track cell proliferation, differentiation, and/orfunction.
 48. A composition for tracking cell proliferation,differentiation, and/or function, the compositions comprising: (a) oneor more of the compounds of claim 1; and (b) an analyte, wherein the oneor more of the compounds are present in an amount effective to trackcell proliferation, differentiation, and/or function. 49-52. (canceled)